Multiple antigen delivery system using hepatitis E virus-like particle

ABSTRACT

This invention provides a peptide/nucleic acid composition for oral/mucosal, dual-modal activation of immune protection systems.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US2010/025803, filedMar. 1, 2010, which claims priority to U.S. Provisional PatentApplication No. 61/156,446, filed Feb. 27, 2009. This application alsoclaims priority to U.S. Provisional Patent Application No. 61/408,501,filed Oct. 29, 2010. The contents of each of the above are herebyincorporated by reference in the entirety for all purposes.

BACKGROUND OF THE INVENTION

HEV is an ss(+)RNA virus causing self-limited hepatitis in human. Whenexpressed in insect Tn5 cells, the truncated capsid protein (CP)covering residues 112-608 is able to self-assemble into virus-likeparticle (VLP). The VLP is smaller than the HEV native virion andcontain no HEV genomic RNA. The VLP itself induces efficiently theimmunity in chimpanzee against the challenge of HEV virus withoutadjuvant. The structure of this HEV VLP has recently been achieved inatomic resolution by the present inventors to elucidate that HEV CP iscomposed of three major domains, S1, S2, and P, with epitopepresentation and capsid assembly modularized in independent modalities(Wang et al., 2008. Acta Crystallographica F, Acta Cryst. F 64, 318-322;Xing et al., 1999. Virology 265, 35-45). HEV VLP is a potential carrierof mucosal vaccine for the presentation of antigenic epitopes throughoral administration. The chimeric CP, with an insertion of 11 aminoacids of a B-cell epitope tag at the C-terminus of a truncated CP, stillforms icosahedral particle. After oral administration, this HEV chimericVLP is able to stimulate humoral immune response and significant levelof IgM and IgG antibodies to the inserted epitope and HEV were observedin intestinal secretions (Niikura et al., 2002. Virology 293, 273-280).Importantly, the HEV VLP can disassemble and reassemble in vitro withthe ability of encapsidating DNA plasmids. With this method, the HEV VLPis demonstrated to deliver DNA plasmid encoding human immunodeficiencyvirus (HIV) gp120 into the intestinal mucosa. Significant level ofspecific IgG and IgA to HIV env was found in fecal extracts and sera oftesting experimental animal. Moreover, mice used in the study exhibitedCTL response specific to gp120 in the spleen, Payer's patches andmesenteric lymph modes. (Takamura, S., et al., 2004. Gene Ther 11,628-63.) In summary, these data demonstrate that the HEV VLP is capableof delivering both amino acid antigens and DNA encoding the antigens toor conferring immunity to mucosal tissue by oral administration.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a composition comprising a modifiedhepatitis E virus (HEV) capsid protein and a heterologous nucleic acidencapsulated in a chimeric virus-like particle (VLP) formed by themodified HEV capsid protein. The modified HEV capsid comprises a portionof HEV ORF 2 protein and a heterologous peptide. In some embodiments,the heterologous nucleic acid and the heterologous peptide are from thesame source. In some embodiments, the heterologous nucleic acid and theheterologous peptide are from different sources. In some embodiments,the modified HEV capsid protein comprises a portion of HEV ORF 2 proteinand two or more heterologous peptides. The two or more heterologouspeptides can be from the same source or from different sources.

In one embodiment of the invention, the modified HEV capsid protein ofthe composition of the present invention comprises residues 112-608 ofHEV ORF 2 protein. For example, the modified HEV capsid comprisesresidues 112-608 of HEV ORF 2 protein and a heterologous peptide. Inanother embodiment of the invention, the modified HEV capsid proteinfurther comprises residues 91-123 of HEV ORF 3 protein fused to theN-terminal of residues 112-608 of the HEV ORF 2 protein. In otherembodiments of the invention, the modified HEV capsid protein furthercomprises residues 70-123 of HEV ORF 3 protein fused to the N-terminalof residues 112-608 of the HEV ORF 2 protein. For instance, a modifiedHEV capsid can also be a fusion protein of residues 112-608 of the HEVORF 2 protein and residues 70-123/91-123 of the HEV ORF 3 protein and aheterologous peptide.

In some embodiments of the invention, the heterologous peptide islocated at the C-terminal of the modified HEV capid protein. In someembodiments of the invention, the heterologous peptide is located at theN-terminal of the modified HEV capid protein.

In some embodiments of the invention, the heterologous peptide isinserted into HEV ORF 2 protein of the modified HEV capid protein withina pre-selected region selected from a group consisting of residues483-490, residues 530-535, residues 554-561, residues 573-577, residues582-593, and residues 601-613. In other embodiments of the invention, atleast one residue of the pre-selected region is deleted. In some cases,all residues within the pre-selected region are deleted.

The compositions of the present invention may further comprise apharmaceutically acceptable excipient. The excipient may be suitable fororal delivery of the composition. Alternatively, the exicipient may beadapted for mucosal delivery of the composition. In some embodiments ofthe invention, the exicipient is an adjuvant.

The present invention further provides a method of inducing animmunogenic response in a host, comprising the step of administering tothe host an effective amount of the composition of this invention.

The present invention also relates to a modified hepatitis E virus (HEV)capsid protein and polynucleotides encoding such protein. The modifiedhepatitis E virus (HEV) capsid protein comprises a portion of HEV ORF 2protein and a heterologous peptide inserted into the portion of HEV ORF2 within a pre-selected region of residues 483-490, residues 530-535,residues 554-561, residues 573-577, residues 582-593, or residues601-613 of the HEV ORF 2 protein.

In an additional aspect, the present invention provides a means fordelivery of therapeutics, especially to various mucosal tissues. Forinstance, the VLPs of this invention may be used to encapsulate anddeliver an agent for therapeutic purpose. The capsid proteins formingthe VLPs may comprise only portions or full length of the HEV proteinsuch as ORF2 or ORF3 or may comprise a portion of a heterologousprotein. The therapeutic agent may be of any chemical nature, includingsmall molecules or macromolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Characterization of VLP/C-tag and VLP/MAb224. (A) Westernblotting assay of the C-terminally truncated ORF2 proteins with MAb224.M, molecular weight markers; W, wild-type baculovirus-infected cells.(B) Diagram of the C-terminal markers. Electron micrographs offrozen-hydrated VLP/C-tag (C) and VLP/MAb224 (D). Both particles show anabsence of density in the center of particles. Note the surface ofVLP/MAb224 showed longer thorn-like densities compared to VLP/C-tag.Scale bar presents 50 nm.

FIG. 2 Surface representation of the HEV structures. (A) VLP/C-tag and(B) VLP/MAb224 viewed along the icosahedral two-fold axis. The positionsof the two-, three-, and five-fold axis are marked as oval, triangle,and pentagon, respectively. The density level was used to account for100% of the expected protein volume. In both reconstruction the capsidprotein, truncated ORF2 protein, establishes an arrangement according toa T=1 surface lattice with 30 protruding spikes at each two-foldpositions. In (B), the MAb224 appeared as two domains configuration,where the variable domain was bound to the side-shoulder of the P domainof VLP, and the constant domain was slightly tilted perpendicular to thesurface of the particle. The scale bar is 100 Å. (C) Resolutionestimated by FSC. The particles of each map were evenly divided into twosub-datasets and reconstructed. The correlation was used as a functionof spatial frequency (1/resolution, A) between two independentreconstructions. The resolution of VLP/C-tag (dashed line) wascalculated to 17.5 Å and the resolution of VLP/MAb224 was calculated to18.5 Å (solid line).

FIG. 3 The difference maps for the tag epitope and MAb224 at thetwo-fold axis. (A) Difference map calculated by subtracting VLP/C-tagfrom VLPTn5, and the resulting C-terminal inserted tag epitope wassuperimposed on the corresponding region of the reconstruction ofVLPTn5, and the density level was chosen to account for 100% mass ofexpected tag volume using the average protein density of 1.36 g/cm3. (B)Difference map calculated by subtracting VLP/MAb224 from VLPSf9, and theresulting MAb224 fragments was superimposed on the corresponding regionof the reconstruction of VLPSf9.

FIG. 4 Secondary and tertiary structure predictions of C-terminalpeptide (SEQ ID NO:3). Alpha-helix was marked with the letter H andbeta-sheet was marked with E. Stereo view of the tertiary structure wasshown in the inset at the right bottom. The protein backbone wasrendered in a ‘cartoon’ style to highlight the secondary structure.

FIG. 5 Fitting of the predicted 3D atomic model into cryo-EM densitymaps. (A and C) top views and (B and D) side views of VLP/C-tag andVLP/MAb224. Cryo-EM density maps (as shown in mesh) were fitted with thepredicted model shown in a cartoon representation. The predicted atomicmodel was manually fitted into the cryo-EM density according to theepitope region and C-terminus region of cryo-EM density maps. Theepitope position was exposed to the surface and located at the side ofthe P domain.

FIG. 6 Surface mapping of the epitope and C-terminus. (A) The predicted3D model fitted in the difference map of MAb224 together with VLPSf9viewed from the side of the P domain. The epitope region is shown in asurface representation. Note the binding site of the neutralizingantibody overlaps with the binding site of MAb224, and the footprint ofMAb224 covers the whole epitope. (B) The C-terminal route started fromthe lateral sides of P domain, which was found interacting withantibodies recognized the antigenic site at the carboxyl terminus, andended at the top surface of the P domain.

FIG. 7 The atomic structure of C-terminal P domain determined by X-raycrystallography. The structure was shown as ‘cartoon’ mode to highlightthe secondary structure. The amino acids 555-560 are disordered in thecapsid and are less resolved from the electron density map.

FIG. 8 HEV-VLP having a chimerized epitope with a DNA vaccineencapsulated.

FIG. 9 Structure of HEV-VLP and capsid protein dimer of HEV-VLP.

FIG. 10. Crystal structure of HEV-LP and comparison of capsid proteindimers of HEV-LP, rNV, SMSV, and CARMV. The S, M, and P domains of theHEV capsid protein are indicated. (A) HEV-LP is composed of sixty capsidsubunits forming icosahedral 2-, 3-, and 5-fold axes and indicating aT=1 symmetry. (B) The ribbon diagram of a capsid subunit of HEV-LP (PDBaccession code: 2ZTN) shows P, M, and S domains at the top, middle, andbottom, respectively. The disordered regions are shown with dashedlines. The S domain shows a jerry roll-like β-barrel structure conservedin some viruses. The conserved anti-parallel β-strands are indicated (Bto I). (C) The ribbon diagrams of crystal structures of capsid proteindimers of HEV-LP and those of rNV (PDB accession code 1IHM), SMSVc(PDBaccession code 2GH8), and CARMV (PDB accession code 1 OPO) areindicated.

FIG. 11. Interaction of capsid protein subunits of HEV-LP around the5-fold axis. (A) The pentamer of the capsid protein of HEV-LP. Theclose-up surface diagram of the 5-fold axis showed from outside andinside of HEV-LP. Amino acid residues Asn-200 and Tyr-288 are shown. Theclose-up surface diagram of the 5-fold axis showed from outside of rNV,SMSV, and CARMV. The aromatic amino acids Phe-118 of rNV, Tyr-330 ofSMSV, and Phe-145 of CARMV are indicated. The deduced interacting atomsare connected with dashed lines, and the distances are indicated. (B)Sucrose density fractionation assay using the wild-type or mutant capsidproteins (53 kDa) in which the amino acids composing the 5-fold axiswere substituted. The capsid protein composing HEV-LP was found in the5-9^(th) fractions from the top, while that which failed to formparticles was found in the top fractions. The molecular mass ofapproximately 64 kDa was a nonspecific protein.

FIG. 12. Characterization of monoclonal antibodies and mutant HEV-LPs.(A) Neutralization of binding (NOB) of HEV-LP to Huh7 cells bymonoclonal antibodies to HEV-LP. After preincubation of HEV-LP (10μg/mL) with each of the monoclonal antibodies (20 μg/mL) for 1 h at 37°C., the mixture was inoculated into Huh7 cells and incubated for 1 h at4° HEV-LP (lined area) bound to cells was detected by flow cytometry.The filled area indicates mock-incubated cells. (B) Construction ofHEV-LP mutants. Sixteen HEV-LP mutants, in which the surface amino acidresidues of the P domain were substituted, were constructed. The proteinbands of 100 ng each of the purified wild-type and mutant HEV-LPs werevisualized by Coomassie brilliant blue staining after SDS/PAGE. (C)Reactivities of NOB antibodies with the mutant HEV-LPs.Immunoprecipitation analyses of a series of HEV-LPs by NOB (MAB1323 andMAB272) or non-NOB antibodies (MAB358 and MAB161). ImmunoprecipitatedHEV-LPs were detected by an anti-HEV capsid rabbit polyclonal antibody.(D) Binding capability of the mutant HEV-LPs to Huh7cells. Wild-type ormutant HEV-LPs (10 m/mL) were incubated with Huh7 cells for 1 h at 4°C., and then HEV-LP (lined area) bound to cells was detected by flowcytometry. The filled area indicates mock-incubated cells. The MFI isshown in each panel.

FIG. 13. Amino acid residues involved in the recognition by NOBantibodies and in the binding to Huh7 cells. Surface diagrams of thecapsid protein dimer from a lateral (Upper) or top (Lower) view. (A)Amino acids in HEV-LP involved in the complete loss or reduction ofreactivity to MAB1323 and MAB272 are shown. (B) Amino acids in HEV-LPresponsible for binding to Huh7 cells are shown. The substitutions inthe P domain of HEV-LP that exhibited no effect on the reactivity withNOB antibodies or the binding to Huh7 cells are shown.

FIG. 14. The cryo-EM structure of HEV T=1 VLP in complexed with the Fabfragments. (A) Surface presentation of VLP/Mab224 (left) and that ofVLP/Mab4 (right) viewed along one of the icosahedral twofold axis. Onefivefold axis and two adjacent threefold axes are marked withcorresponding number. In both reconstructions, sixty copies of Fabfragment attached to the later side of HEV VLP; however, the density ofMab4 Fab fragment appears weaker than that of Mab224 Fab fragment. (B)The viral surface is shown as a stereographic projection overlapped withline drawn an icosahedral asymmetric unit. The fivefold, threefold andtwofold axis are marked with corresponding numbers. The Fab density isprojected as white circles on the viral surface.

FIG. 15. The binding site of Mab224 antibody. (A) The cryo-EM densitymap of VLP/Mab224 was fitted with the crystal structure of PORF2 andviewed along a bound Fab molecule. One PORF2 dimer is presented as solidsurface and the neighboring dimers are drawn as ribbon mode. (B) Theside view of a PORF2 dimer fitted into the cryo-EM density map. (C) APORF2 dimer viewed along the twofold axis overlapped with the cryo-EMdensity map. (D) The top view of a PORF2 dimer viewed along the twofoldaxis. The amino acids in PORF2 responsible for binding to Mab224 arelabeled. The PORF2 dimer is presented as solid surface.

FIG. 16. The structure of the chimeric HEV VLP carrying a B-cell tag.(A) Surface presentation of VLP/C-tag viewed along an icosahedraltwofold axis. (B) The cryo-EM density map of VLP/C-tag (mesh) was fittedwith the crystal structure of PORF2 decamer (Ribbon). (C) Ribbonrepresentation of PORF2 dimer. The four selected internal insertionsites are marked as sphere mode representing the element. (D) The topview of the PORF2 dimer showing the location of the internal insertionsites.

FIG. 17. Location of the C-terminal insertion relative to the Mab224binding site. The side view (A) and the top view (B) of the fitted PORF2dimer (surface presentation) overlapped the cryo-EM density map ofVLP/C-tag (mesh). The C-terminal residue A606 is marked. The ribbonpresentation shows the adjacent dimers.

FIG. 18. The HEV large VLP is composed of 180 copies of ORF2 protein.(A) STEM micrograph of HEV-VLPs. Both large and small T=1 HEV-VLPs areprojected as spherical images and their corresponding particle mass wascalculated. The long straight rod is tobacco mosaic virus (TMV), whichwas added as an internal mass standard. (Bar=1000 Å). (B) The largeHEV-VLP appeared as an intact particle decorated with a dot-like patternon the surface on cryo-EM of the large HEV-VLP. (Bar=500 Å). (C) Massspectrum of the large HEV-VLP showing that the molecular mass of theORF2 protein is 65.5 KD. (D) Plot showing the observed mass/length ofTMV against the observed mass of the large HEV-VLP from different imageconditions. For a known TMV mass/length of 13.1 kD/Å, and the mass ofHEV large VLP was calculated as 11.8 MDa.

FIG. 19. Three-dimensional structure of HEV T=3 VLP. (A) Overallstructure of HEV large VLP reveals the T=3 icosahedral lattice of theORF2 proteins. One icosahedral facet is defined as the triangular areawithin the three adjacent fivefold axes. (B) There are two uniquedimeric ORF2 spikes on the HEV T=3 VLP surface. The AB dimer is locatedaround the fivefold axis and the CC dimer is located at the twofoldaxis. (C) HEV T=3 VLP has a radius of 205 Å and contains a low densitycavity with a radius of 85 Å in the particle center. The distribution ofthe cryo-EM density revealed four ORF2 domains, P, S2, S1, and N, at 50Å from the equatorial section. (D) The crystal structure of the HEVsubunit from T=1 VLP docks well with the cryo-EM density in the shellregion of HEV T=3 VLP, with the N-terminal loop pointing towards thecenter.

FIG. 20. The structure of genotype-1 PORF2 protein. (A) Ribbonrepresentations of S1, S2, and P. (B) Surface areas that buried at theinterfaces between two adjacent subunits are overlapped with a PORF2hexamer (left) and the BSA at the PORF2 dimeric interface (right).

FIG. 21. Structure of ORF2 decamer is consistent in both T=1 and T=3VLPs. (A) The cryo-EM density of HEV T=3 VLP agrees well with thecoordinates of T=1 VLP at the region around the fivefold axis. The P,S2, S1, and N+RNA mark the corresponsding density layer. (B) Consistentfeatures between the decamer in HEV T=3 and T=1 VLP were revealed by thecrystal structure docking of PORF2 dimers into the cryo-EM density mapof HEV T=3 VLP. (C) Reassembly of the ORF2 protein in vitro led to ORF2star-shaped complex formation (white arrows). The size of this complexfits well into that of the ORF2 decamer after calibration with TMV(black arrow) as an internal standard. One ORF2 complex was zoomed intwice and is displayed as an inset in comparison with the crystalstructure of PORF2 pentamer (ribbon drawing). (D) An RNA density wasextracted from HEV T=3 VLP (lane B) but not T=1 VLP (lane A). The RNAsize ladder was loaded on lane M.

FIG. 22. The orientation of P domain relative to the S2 and S1 domain inthe C-C dimer appears different to that in A-B dimer. (A) The dimericinteractions in the HEV A-B dimer, the HEV C/C dimers and the Norwalkvirus C-C dimer are shown in CPK models as observed from the outside ofthe particles. (B) In the T=3 cryoEM density map, the orientation of theP domain is shown as the angle between the platform of the spikerelative to the S2/S1 domains in the C-C dimer (white line passingthrough the two adjacent threefold axes) or the S2/S1 domains in the A-Bdimer (while line passing through a fivefold axis and the neighboringthreefold axis). (C) The crystal density map showing the position of theproline-rich hinge within the cleft of the S2 domains. (D) The cleft inbetween the S2/S1 domains provides sufficient room to accommodate theproline-rich hinge in the A-B dimer, where the domains taking bentconformation. The cleft is narrow down in the C-C dimer due to the flatconformation between the S2/S1 domains thus push up the hinge out of thecleft.

FIG. 23. Diagram showing the putative assembly process of HEV T=1 andT=3 VLP. The ORF2 subunit encodes information that governs the assemblyof decamers. Interaction with RNA fragment induces flat dimeric contactand the formation of C-C dimers, which guides the assembly of completeicosahedral capsid.

FIG. 24. Comparison between HEV capsid proteins of genotype-1, -3, and-4. (a) Sequence alignment of HEV genotypes-1, -3, and -4 (SEQ IDNOS:4-6). Amino acid residues are boxed according to the alignmentgenerated by CLUSTAL-X. Secondary structural elements are labeled abovethe sequence. (b) Ribbon representations of the monomer structures ofthe HEV-VLP PORF2 protein of genotype-1, genotype-3 and genotype-4. (c)Structures of HEV PORF2 proteins after a 90° rotation to show thelocations of the N-termini.

FIG. 25: Structure of HEV S2 and P domain and their difference fromcalciviruses. (a) Ribbon representations (Left) of the S2 domain in HEVand the P1 domain in SMSV along with their respective topology diagrams(Right). The β-strands are labeled from A′ to F′. (b) Ribbonrepresentations (Left) of the P and P2 domains in HEV, SMSV and NV alongwith their respective topology diagrams (Right). The β-strands arelabeled from A″ to F″, following the nomenclature of SMSV.

FIG. 26. Technical data for cryo-EM reconstruction of a large HEV-VLP.(a) Distribution of cryo-EM with a defocus level of 0.7-3.5 μm. (b)Fourier shell correlation indicating that the resolution of the finaldensity map is 10.6 Å (cutoff of 0.5).

FIG. 27. Stereoimage of the T=1 VLP electron density map with modeledamino acid residues in the S1 domain.

FIG. 28. Overall strucuture of genotype 1 HEV-VLP. (a) A CPK model ofthe capsid structure viewed down an icosahedral two-fold axis. The whitetriangle circulates an icosahedral face. Bar=100 Å. (b) Ribbonrepresentations of the dimer structure of the HEV-VLP capsid protein.(c) A ribbon representation of the Si domain. The β-strands are labeledfrom B to I, following the nomenclature of caliciviruses. (d) A ribbonrepresentations of the S2 domain. The β-strands are labeled from A′ toF′. (e) A ribbon representations of the P domain. The β-strands arelabeled from A″ to F″.

FIG. 29. ORF2 Intersubunit interactions. (a) Surface areas that buriedat the interface between the subunits are shown. The white trianglecirculates the area of an icosahedral face. (b) A ribbon representationshowing the contact at icosahedral fivefold with five Y288 highlightenas stick mode. (c) P domain dimeric interface. The surface electronpotential of one P domain is shown. The interacting hydrophobic aminoacids from the other subunit are shown as sticks. (d) The density voidedchannel at threefold position. The surface electron potential of twothreefold-related subunits is shown at background to reveal the innersurface. The sticks shows the critical negatively charged amino acids ofthe third subunit around threefold axis.

FIG. 30. HEV-VLP structure demonstrates its cargo ability for deliveryof heterogenous epitopes. (a) Surface representation of a dimer and thesurrounding dimers showing the specific residues for the HEV genotype 1(G1) among HEV genotypes (G1, G3 and G4) as viewed from the outside ofthe shell. The specific residues in the S2 and P domains of the dimerare labeled. (b) Previous insertion sites are failed because theyinterfere with either dimeric or dimer-dimer interactions. Two insertionsites are located at 51 domain and S2 domain respectively. Two othersites are located at dimeric interface of the P domain. (c) Humoralresponse to the chimeric epitope. Mice were given three oralimmunizations with one of the following: chimeric HEV-VLP carrying HIVP18 epitope (P-VLP/P18, square), chimerically-synthesized HIV P18epitopes peptide (triangles), or HEV-VLP (circle). No treatment controlsare shown by diamonds. Sera and fecal suspension (200 mg/ml) werediluted (1:100 and 1:2 respectively) and the specific antibodies to thechimeric epitope were evaluated in ELISA using synthetic peptide as theantigen.

FIG. 31. HEV-VLP is capable of carrying DNA plasmid for gene delivery.(a) A sample electron density map with modeled amino acid residues inthe 51 domain. (b) Electron potentials on the surface of the dimer atthe icosahedral 2-fold axis, as viewed from the inside of the capsidshell. Positively charged residues around 2-fold axis are labeled. Thefigure was prepared with Swiss-PDB Viewer. (c), (d) CTL response to thechimeric epitope and encapsulated DNA vaccine. Mice were orallyimmunized with chimeric HEV-VLP that bore HIV P18 epitope one thesurface and an encapsulated DNA vaccine expressing entire Gag protein(P-P18/N-Gag capsule). Specific lysis by CTL in response to either theP18 epitope (c) or Gag protein (d) was evaluated by testing spleen,mesenteric lymph nodes and Peyer's patch cells from immunized animals(closed circles). Control tissues (open circles) were from mice fed withchemically-synthesized P18 epitope peptide (c) or a naked DNA vaccineexpressing Gag (d). The effector/target ratios (80/1, 40/1, and 20/1,shown left to right in each panel) were tested.

FIG. 32: Separation of DNA-loaded HEV-VLPs from the empty VLPs by CsC1density gradient.

FIG. 33: Level of anti-P18 specific IgG (a, c) and IgA (c, d) antibodiesin sera (a, b) and fecal extrat (c, d).

FIG. 34: Spleen, MLN, and PP cells from mice orally receivedplasmid-loaded VLP (circles) elicited specific CTL responses, while thenaked plasmid (triangles) and non-immunized control (cross) did not.

FIG. 35: Characterization of VLP/C-tag and VLP/Fab224. (A) Westernblotting assay of the C terminally truncated ORF2 proteins with Fab224.M: molecular weight markers; W: peptides recovered frombaculovirus-infected cells. (B) Diagram of the C-terminal markers. (C)Electron micrograph of frozen-hydrated VLP/C-tag and (D) micrograph offrozen-hydrated VLP/Fab224. Black arrows indicate the Fab moleculesattached to the VLP. Both particles showed an absence of density in thecenter. Note the surface spike in VLP/Fab224 appeared as longerthorn-like densities compared to VLP/C-tag. Scale bar presents 50 nm.

FIG. 36: The cryo-EM structure of HEV T=1 VLP in complex with anti-HEVantibodies. (A) Surface presentation of VLP/Fab224 (left) and that ofVLP/Fab4 (right) viewed along one of the icosahedral twofold axes. Onefivefold axis and two adjacent threefold axes are marked with thecorresponding number. In both reconstructions, 60 copies of Fab areattached to the lateral side of HEV VLP; however, the density of Fab4molecule appears weaker than that of Fab224 molecule. (B) The viralsurface is shown as a stereographic projection overlapped with a linedrawing an icosahedral asymmetric unit. The fivefold, threefold andtwofold axes are marked with corresponding numbers, while the blacktriangle encloses the area of an icosahedral asymmetric unit. Thesurface residues are colored according to the distance from the centerof the VLP, with red being the furthest away and blue representing thesurface depressions. The Fab density is projected as white circles onthe viral surface and the most outer density-layer was drawn as thickwhite circles.

FIG. 37: The binding site of Fab224 antibody. (A) The cryo-EM densitymap of VLP/Fab224 was fitted with the crystal structure of PORF2 andviewed along a bound Fab molecule. One PORF2 dimer is presented as solidsurface and colored as light magenta for S-domain, slate for M-domainand dark grey for P-domain. The neighboring dimers are drawn as ribbonmode and colored wheat. (B) The side view of a PORF2 dimer fitted intothe cryo-EM density map. (C) A PORF2 dimer viewed along the twofold axisoverlapped with the cryo-EM density map. (D) The top view of a PORF2dimer viewed along the twofold axis. The amino acids in PORF2responsible for binding to Fab224 are labeled. The PORF2 dimer ispresented as solid surfaceand colored in gray, slate, light magenta forthe P domain, the M-domain and the S-domain, respectively. The residuesalong the Fab binding interface are colored according to the elementwith green for carbon, blue for nitrogen and red for oxygen.

FIG. 38: The structure of the chimeric HEV VLP carrying a B-cell tag.(A) Surface presentation of VLP/C-tag viewed along an icosahedraltwofold axis. (B) The cryo-EM density map of VLP/C-tag (mesh) was fittedwith the crystal structure of PORF2 decamer (Ribbon). (C) Ribbonrepresentation of PORF2 dimer with one monomer colored as gray and theother colored as pink for 51 domain, blue for M-domain and lime for theP domain. The amino acids prior to the four internal insertion sites aremarked in sphere mode with color code representing the element. (D) Thetop view of the PORF2 dimer showing the location of the non-VLPinsertion sites.

FIG. 39: Fitting of the PORF2 structure into the cryo-EM density map ofHEV VLP/C-tag. The side view (A) and the top view (B) of the fittedPORF2 dimer (surface presentation) overlapped with the cryo-EM densitymap of VLP/C-tag (mesh). The C-terminal residue A606 is located at theside of the protruding spike. One PORF2 dimer is colored as lightmagenta, slate, and gray for the S-, M- and P-domain on the surfacepresentation, respectively. The ribbon representation shows the adjacentdimers. The amino acids in PORF2 responsible for binding to Fab224 werecolored in green for carbon, blue for nitrogen, and red for oxygen.Asterisks mark the location of the extra density that was not occupiedwith PORF2 coordinates.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention relates to an HEV VLP-based peptide/nucleic acid(P/N) system. The idea of using HEV VLP-based P/N system providesseveral unique advantages, besides inducing immune response via both MHCclass I and II system. 1) the P/N system provides the possibility oforal vaccination. Practically, oral administration is less stressful anddoes not require professional skill. Besides, delivery of vaccinethrough intestinal tract is considered safer than systemic injection.The nature infection route of HEV and the structure of HEV VLP provideresistance to hash environments in the digestive tracts, such as low pHin the stomach, the presence of proteolytic enzymes, and the presence ofphysical and biochemical barriers associated with the mucosal surface.2) the HEV VLP can be produced from standard cultivation protocols andthe yield of purified HEV-VLPs can be 50-100 μg/ml, about 100 timesgreater compared to other VLPs. 3) the HEV VLP deliver amino acidimmunogen in the form of icosahedral particles, ie, every successfullyentered particle brings in 60 copies of immunogen to the same host cell.4) anti-HEV immune responses are proven to have no effect on both DNAadministration and peptide immunogen vaccination. 5) the HEV is stableat room temperature. By combining all the features, we thereforeanticipate the P/N system would accomplish our goal of developefficacious and broadly reactive vaccine.

II. Definitions

“Hepatitis E virus” or “HEV” refers to a virus, virus type, or virusclass, which i) causes water-borne, infectious hepatitis; ii)distinguishes from hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), or hepatitis D virus (HDV) in terms ofserological characteristics; iii) contains a genomic region that ishomologous to a 1.33 kb cDNA inserted in pTZKF1(ET1.1), a plasmidembodied in a E. coli strain deposited in American Type CultureCollection (ATCC) with accession number 67717.

The terms “capsid protein” or “modified capsid protein”, with referenceto HEV, refer to a mature or modified ORF2 or ORF 3 polypeptide. As usedherein, reference to such ORF 2 or ORF 3 polypeptides or proteins ismeant to include the full-length polypeptide, and fragments thereof, andalso include any substitutions, deletions, or insertions or othermodifications made to the ORF 2 or ORF 3 proteins.

As used herein, the term “virus-like particle” (VLP) refers to astructure that in at least one attribute resembles a virus but is notinfectious due to the lack of a viral genome. “VLP” refers to anonreplicating viral shell, preferably derived from hepatitis E virusproteins such as capsid proteins. VLPs are generally composed of one ormore viral proteins, including, but are not limited to those proteinsreferred to as Hepatitis E capsid proteins, or modified hepatitis Evirus capsid protein. VLPs can form spontaneously upon recombinantexpression of the protein in an appropriate expression system.

The term “heterologous nucleic acid,” as used herein, refers to anucleic acid not endogenous to the hepatitis E virus, i.e., from asource other than HEV. The term “heterologous polypeptide,” as usedherein, refers to a peptide or polypeptide not endogenous to thehepatitis E virus, or a peptide or polypeptide to a protein or peptidecoded for by a DNA sequence which is not endogenous to the native genomeof hepatitis E virus, i.e., from a source or organism other than HEV.

The term “encapsulation,” or “encapsulated,” as used herein refers tothe envelopment of a heterologous substance, such as a heterologousnucleic acid, within the virus-like particles defined herein.

The term “chimeric protein” refers to an amino acid sequence having twoor more parts that generally are not found together in an amino acidsequence in nature. The term “chimeric virus-like particle” refers to avirus-like particle comprising HEV capsid proteins and one or moreheterologous peptide. As defined herein, the term “chimeric virus-likeparticle” further refers to a virus-like particle comprising an HEVcapsid protein and a heterologous peptide encapsulating one or moreheterologous nucleic acids.

As defined herein, the term “source” refers to a pathogen. The term“source” may also refers to the cells derived from diseased tissues,e.g., tissues of a cancer, infectious disease, allergic reaction, orautoimmune disease. Pathogens include, for example, a bacterium, virus,protozoan, fungus, parasite, or infectious particle, such as a prion.Examples of pathogens further include Adenoviradae; Arenaviridae (forexample, Ippy virus and Lassa virus); Birnaviridae; Bunyaviridae;Caliciviridae; Coronaviridae; Filoviridae; Flaviviridae (for example,yellow fever virus, dengue fever virus and hepatitis C virus);Hepadnaviradae (for example, hepatitis B virus); Herpesviradae (forexample, human herpes simplex virus 1); Orthomyxoviridae (for example,influenza virus A, B and C); Paramyxoviridae (for example, mumps virus,measles virus and respiratory syncytial virus); Picornaviridae (forexample, poliovirus and hepatitis A virus); Poxviridae; Reoviridae;Retroviradae (for example, BLV-HTLV retrovirus, HIV-I, HIV-2, bovineimmunodeficiency virus and feline immunodeficiency virus);Rhabodoviridae (for example, rabies virus), and Togaviridae (forexample, rubella virus). In one embodiment, the products comprise one ormore antigens derived from a major viral pathogen such as the varioushepatitis viruses, polio virus, human immunodeficiency virus (HIV),various influenza viruses, West Nile virus, respiratory syncytial virus,rabies virus, human papilloma virus (HPV), Epstein Barr virus (EBV),polyoma virus, or SARS coronavirus. Specific examples of hepatitisviruses include, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), delta hepatitis virus (HDV), hepatitis E virus(HEV) and hepatitis G virus (HGV); Specific examples of herpesvirusfamily include herpes simplex virus (HSV) types 1 and 2. Pathogensfurther include agents causing diseases such as diptheria (e.g.,Corynebacterium diphtherial), pertussis (e.g., Bordetella pertussis),tetanus (e.g., Clostridium tetan?), tuberculosis (e.g., Mycobacteriumtuberculosis), bacterial or fungal pneumonia, cholera (e.g., Vibriocholerae), typhoid fever (e.g., S. typhi), plague, shigellosis (e.g.,Shigella dysenteriae serotype 1 (S. dysenteriae I)), Salmonellosis,Legionnaire's disease (e.g., Legionella pneumophila), Lyme disease,leprosy (e.g., Mycobacterium leprae), malaria (e.g., Plasmodiumfalciparum), Hookworm, Onchocerciasis, Schistosomiasis,Trypamasomialsis, leishmaniasis, giardia (e.g., Giardia lamblia),Amoebiasis (e.g., Entamoeba histolytica), Filariasis, Borrelia,Trichinosis, influenza, hepatitis B and C, meningococcal meningitis,community acquired pneumonia, chickenpox, rubella, mumps, measles, AIDS,dengue respiratory infections, diarrhoeal diseases, tropical parasiticdiseases, sexually transmitted diseases and chlamydia infections.Antigenic material may also be derived from causative agents responsiblefor new emerging, re-emerging diseases or bioterrorism diseases such as:SARS infection, Vancomycin-resistant S. aureus infections, West NileVirus infections, Cryptosporidiosis, Hanta virus infections, EpsteinBarr virus infections, Cytomegalovirus infections, H5N1 influenza,Enterovirus 71 infections, E coli. O157:H7 infections, human monkey pox,Lyme disease, Cyclosporiasis, Hendra virus infections, Nipah virusinfections, Rift Valley fever, Marburg haemorrhagic fever, Whitewaterarrollo virus infections and Anthrax.

As defined herein, the term “same source” refers to the fact that aheterologous nucleic acid and a heterologous peptide (or two or moreheterologous peptides) are derived from the same organism, such as adisease-causing pathogen including virus, bacterium, etc. The “samesource” encompasses mutated or modified forms of the pathogen, such asdifferent strains of a virus or bacterium.

A “pharmaceutically acceptable” or “pharmacologically acceptable”material is one that is not biologically harmful or otherwiseundesirable, i.e., the material may be administered to an individualalong with the modified hepatitis E virus capsid protein or the chimericvirus-like particles or the compositions of the present inventionwithout causing any undesirable biological effects. Neither would thematerial interact in a deleterious manner with any of the components ofthe composition in which it is contained.

The term “excipient” refers to any essentially accessory substance thatmay be present in the finished dosage form of the composition of thisinvention. For example, the term “excipient” includes vehicles, binders,disintegrants, fillers (diluents), lubricants, glidants (flowenhancers), compression aids, colors, sweeteners, preservatives,suspending/dispersing agents, film formers/coatings, flavors andprinting inks.

The term “adjuvant” refers to a compound that, when administered inconjunction with an antigen, augments the immune response to theantigen, but does not generate an immune response to the antigen whenadministered alone. Adjuvants can augment an immune response by severalmechanism including lymphocyte recruitment, stimulation of B and/or Tcells, and stimulation of macrophages.

An “immunogenic response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present disclosure, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen- specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies by B-cells; and/or the activation of suppressorT-cells and/or γΔ T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

As used herein, the term “host” refers to humans as well as otheranimals.

The term “mucosal delivery” relates to delivery of a composition to amucous membrane, such as the mucosa of the gastro-intestinal tract(e.g., the buccal or labial mucosa) or the mucosa of the respiratorytract (e.g., the nasal mucosa).

III. Hepatitis E Virus Capsid Protein

Hepatitis E virus (HEV) is known to cause severe acute liver failure.HEV belongs to the genus Hepevirus in the family Hepeviridae. HEVcontains a single-stranded positive-sense RNA molecule of approximately7.2-kb. The RNA is 3′ polyadenylated and includes three open readingframes (ORF). ORF1 encodes viral nonstructural proteins, located in the5′ half of the genome. ORF2 encodes a protein-forming viral capsid,located at the 3′ terminus of the genome. ORF3 encodes a 13.5-kDaprotein, overlapped with C-terminus of ORF1 and N-terminus of ORF2. ORF3is associated with the membrane as well as with the cytoskeletonfraction.

III. Virus-Like Particles (Vlps)

One aspect of the invention relates to construction of HEV capsidprotein for self-assembly into virus-like particles (VLPs). Variousconstructs of capsid protein can be used for formation of VLPs(Expression and self-assembly of empty virus-like particles of hepatitisE virus. Li TC, Yamakawa Y, Suzuki K, Tatsumi M, Razak M A, Uchida T,Takeda N, Miyamura T., J Virol. 1997 October; 71(10):7207-13. Essentialelements of the capsid protein for self-assembly into empty virus-likeparticles of hepatitis E virus. Li T C, Takeda N, Miyamura T, MatsuuraY, Wang J C, Engvall H, Hammar L, Xing L, Cheng R H. J Virol. 2005October; 79(20):12999-3006.). An HEV capsid protein comprising HEV ORF 2protein can be used as a construct for formation of VLPs in vitro, sinceHEV's major capsid protein is encoded by ORF 2 gene. Preferably, an HEVcapsid protein comprising a portion of HEV ORF 2 protein can be used asa construct for formation of VLPs in vitro. Optionally, an HEV capsidprotein comprising a portion of HEV ORF 2 protein and a portion of HEVORF 3 protein can be used as a construct for formation of VLPs in vitro.HEV ORF 2 is a protein of 660 residues having the following amino acidsequence:

(SEQ ID NO: 1; GenBank Accession No: AAA45736.1)MRPRP ILLLL LMFLP MLPAP PPGQP SGRRR GRRSG GSGGG FWGDR VDSQPFAIPYI HPTNP FAPDV TAAAG AGPRV RQPAR PLGSA WRDQA QRPAV ASRRRPTTAG AAPLT AVAPA HDTPP VPDVD SRGAI LRRQY NLSTS PLTSS VATGT NLVLYAAPLS PLLPL QDGTN THIMA TEASN YAQYR VARAT IRYRP LVPNA VGGYA ISISFWPQTT TTPTS VDMNS ITSTD VRILV QPGIA SELVI PSERL HYRNQ GWRSV ETSGVAEEEA TSGLV MLCIH GSLVN SYTNT PYTGA LGLLD FALEL EFRNL TPGNT NTRVSRYSST ARHRL RRGAD GTAEL TTTAA TRFMK DLYFT STNGV GEIGR GIALT LFNLADTLLG GLPTE LISSA GGQLF YSRPV VSANG EPTVK LYTSV ENAQQ DKGIA IPHDIDLGES RVVIQ DYDNQ HEQDR PTPSP APSRP FSVLR ANDVL WLSLT AAEYD QSTYGSSTGP VYVSD SVTLV NVATG AQAVA RSLDW TKVTL DGRPL STIQQ YSKTF FVLPLRGKLS FWEAG TTKAG YPYNY NTTAS DQLLV ENAAG HRVAI STYTT SLGAG PVSISAVAVL APHSA LALLE DTLDY PARAH TFDDF CPECR PLGLQ GCAFQ STVAE LQRLKMKVGK TREL

Some constructs of the invention are fusion proteins of a portion of HEVORF 2 protein and a heterologous peptide. Some constructs of theinvention are HEV capsid proteins comprising HEV ORF 2 protein withdeletions at the N-terminal region. Some novel constructs of theinvention are HEV capsid proteins comprising HEV ORF 2 proteins having adeletion of at least contiguous 10 amino acids at the N-terminal region.Some novel constructs of the invention are HEV capsid proteinscomprising HEV ORF 2 protein having a deletion of at least contiguous 25amino acids at the N-terminal region, preferably having a deletion of atleast contiguous 50 amino acids, and particularly preferably having adeletion of at least contiguous 100 amino acids. Preferred constructs ofthe invention are HEV capsid proteins comprising HEV ORF 2 proteinshaving a deletion of 111 to 124 residues at the N-terminal region.Particularly preferred constructs of the invention are HEV capsidproteins comprising HEV ORF 2 proteins having a deletions of 1-111 atthe N-terminal region.

Some constructs of the invention are fusion proteins of a portion of HEVORF 2 protein and a heterologous peptide. Some constructs of theinvention are HEV capsid proteins comprising HEV ORF 2 protein withdeletions at the C-terminal region. Some novel constructs of theinvention are HEV capsid proteins comprising HEV ORF 2 proteins having adeletion of at least contiguous 10 amino acids at the C-terminal region.Some novel constructs of the invention are HEV capsid proteinscomprising HEV ORF 2 protein having a deletion of at least contiguous 20amino acids at the C-terminal region, preferably having a deletion of atleast contiguous 30 amino acids, and particularly preferably having adeletion of at least contiguous 50 amino acids. Preferred constructs ofthe invention are HEV capsid proteins comprising HEV ORF 2 proteinshaving a deletion of 52 to 60 residues at the C-terminal region.Particularly preferred constructs of the invention are HEV capsidproteins comprising an HEV ORF 2 protein having a deletion of 609-660,601-660, 602-660, 603-660, 604-660, 605-660, 606-660, 607-660, 608-660,or 609-660 at the C-terminal region.

Some constructs of the invention are fusion proteins of a portion of HEVORF 2 protein and a heterologous peptide. Some constructs of theinvention are HEV capsid proteins comprising HEV ORF 2 protein withdeletions at both the N-terminal region and the C-terminal region. Somenovel constructs of the invention are HEV capsid proteins comprising HEVORF 2 proteins having a deletion of at least contiguous 10 amino acidsat the N-terminal region and a deletion of at least contiguous 10 aminoacids at the C-terminal region. Some novel constructs of the inventionare HEV capsid proteins comprising HEV ORF 2 proteins having a deletionof at least contiguous 25 amino acids at the N-terminal region and adeletion of at least contiguous 20 amino acids at the C-terminal region.Some novel constructs of the invention are HEV capsid proteinscomprising HEV ORF 2 proteins having a deletion of at least contiguous50 amino acids at the N-terminal region and a deletion of at leastcontiguous 30 amino acids at the C-terminal region. Some novelconstructs of the invention are HEV capsid proteins comprising HEV ORF 2proteins having a deletion of at least contiguous 100 amino acids at theN-terminal region and a deletion of at least contiguous 50 amino acidsat the C-terminal region. Preferred constructs of the invention are HEVcapsid proteins comprising HEV ORF 2 proteins having a deletion of 111to 124 residues at the N-terminal region and a deletion of 52 to 60residues at the C-terminal region. Particularly preferred constructs ofthe invention are HEV capsid proteins comprising residues 112-608 of HEVORF 2.

Another aspect of the invention relates to the construction of HEVcapsid protein for self-assembly into virus-like particles (VLPs), usinga portion of HEV ORF 3 protein fused to the N-terminal of a portion ofHEV ORF 2 protein. HEV ORF 3 is a protein of 123 residues having thefollowing amino acid sequence:

(SEQ ID NO: 2; GenBank Accession No: AAA45726.1)MNNMS FAAPM GSRPC ALGLF CCCSS CFCLC CPRHR PVSRL AAVVGGAAAV PAVVS GVTGL ILSPS QSPIF IQPTP SPPMS PLRPG LDLVF ANPPD HSAPLGVTRP SAPPL PHVVD LPQLG PRR

According to the present invention, a portion of HEV ORF 3 can be fusedto the N-terminal of any HEV ORF 2 construct described above. HEV ORF 3fusion useful for the present invention comprises the C-terminal regionof HEV ORF 2, including the dimerization essential region of residues81-123 of ORF 3. Some novel constructs of the invention are HEV capsidproteins comprising at least 60 residues of the C-terminal of HEV ORF 3protein fused to the N-terminal of a portion of HEV ORF 2 protein. Somenovel constructs of the invention are HEV capsid proteins comprising atleast 70 residues of the C-terminal of HEV ORF 3 protein fused to theN-terminal of a portion of HEV ORF 2 protein, preferably comprising atleast 80 residues of the C-terminal of HEV ORF 3 protein fused to theN-terminal of a portion of HEV ORF 2 protein, and particularlypreferably comprising at least 90 residues of the C-terminal of HEV ORF3 protein fused to the N-terminal of a portion of HEV ORF 2 protein.Preferred constructs of the invention are HEV capsid proteins comprisingresidues 91-123 of HEV ORF 3 protein fused to the N-terminal of aportion of HEV ORF 2 protein. Particularly preferred constructs of theinvention are HEV capsid proteins comprising residues 70-123 of HEV ORF3 protein fused to the N-terminal of a portion of HEV ORF 2 protein.

IV. Chimeric Recombinant Hev Virus-Like Particles

One aspect of the invention relates to a modified hepatitis E viruscapsid protein for self-assembly into virus-like particles (VLPs) usingthe various constructs of capsid protein fused with a heterologouspeptide as described in section III above.

In some embodiments of the invention, the heterologous peptide islocated at the C-terminal of the modified HEV capsid protein. In someembodiments of the invention, the heterologous peptide is located at theN-terminal of the modified HEV capsid protein. Preferably the insertionof the heterologous peptide at the C-terminal or the N-terminal of themodified HEV capsid protein does not disrupt the self-assembly of theVLP. More preferably, the inserted heterologous peptide is exposed tothe surface of the VLP for presentation of the heterologus peptide as anantigenic epitope. The length of the heterologous peptide is preferablychosen to be compatible with the formation of VLP. In general, theheterologous peptide can be 3 or 4 amino acids in length, moretypically, 5,6, or 7 amino acids in length, more typically 8 or 9 aminoacids in length, and even more typically 10 or more amino acids inlength.

In other embodiments of the invention, the heterologous peptide of thepresent invention can also be inserted into HEV ORF 2 protein of themodified HEV capsid protein within a pre-selected region. Any regionwithin HEV ORF 2 protein of the modified HEV capsid protein can beselected for insertion of the heterologous peptide. Preferably theinsertion of the heterologous peptide at the pre-selected region of themodified HEV capsid protein does not disrupt the self-assembly of theVLP. More preferably, the inserted heterologous peptide is exposed tothe surface of the VLP for presentation of the heterologus peptide as anantigenic epitope. Particularly preferably, the pre-selected region is aloop region of HEV ORF 2, wherein the loop region is exposed on thesurface of the VLP. Most preferably, the pre-selected region is one ofthe following loop regions: residues 483-490, residues 530-535, residues554-561, residues 573-577, residues 582-593, and residues 601-613. Thelength of the heterologous peptide is preferably chosen to be compatiblewith the formation of VLP. In general, the heterologous peptide can be 3or 4 amino acids in length, more typically, 5,6, or 7 amino acids inlength, more typically 8 or 9 amino acids in length, and even moretypically 10 or more amino acids in length.

When the heterologous peptide is inserted into a pre-selected region ofHEV ORF 2 protein of the modified HEV capsid protein, deletions withinthe pre-selected region can be made to accommodate the insertion of theheterologous peptide. Necessary deletions of the pre-selected region canbe made to maintain the folding of the capsid protein, to facilitate theself-assembly of the VLP, and to maintain or enhance the stability ofthe VLP. Necessary deletions can also be made to allow longerheterologous insertions, particularly in cases of presenting aconformational epitope.

In some embodiments of the invention, at least one residue of thepre-selected region is deleted. In other embodiments of the invention,all residues of the pre-selected region are deleted. In preferredembodiments of the invention, the number of the residues deleted fromthe pre-selected region is the same or about the same as the number ofthe residues of the inserted heterologous peptide.

A skilled artisan would readily recognize that more than one antigen canbe made by incorporating more than one heterologous peptides in themodified HEV capsid protein. For example, a first heterologous peptidemay be inserted at the C-terminal of the modified HEV capsid protein,and a second heterologous peptide may be inserted into a pre-selectedregion.

The present invention also provide a polynucleotide encoding themodified HEV capsid protein or polypeptide as described herein.

V. Production And Purification Of Virus-Like Particles

One aspect of the invention relates to methods for production andpurification of virus-like particles (See, Expression and self-assemblyof empty virus-like particles of hepatitis E virus. Li T C, Yamakawa Y,Suzuki K, Tatsumi M, Razak M A, Uchida T, Takeda N, Miyamura T., JVirol. 1997 October; 71(10):7207-13. Essential elements of the capsidprotein for self-assembly into empty virus-like particles of hepatitis Evirus. Li T C, Takeda N, Miyamura T, Matsuura Y, Wang J C, Engvall H,Hammar L, Xing L, Cheng R H. J Virol. 2005 October; 79(20):12999-3006.Niikura M et al, Chimeric recombinant hepatitis E virus-like particlesas an oral vaccine vehicle presenting foreign epitopes. Virology 2002;293: 273-280). Various expression systems can be used to express themodified hepatitis E virus capsid protein of the present invention.Examples of expression systems useful for the production of virus-likeparticles of the present invention include, but are not limited to,bacterial expression system (e.g., Escherichia coli), insect cells,yeast cells and mammalian cells. Preferred expression system of thepresent invention includes baculovirus expression systems using insectcells. General methods, for example, for handling and preparingbaculovirus vectors and baculoviral DNA, as well as insect cell cultureprocedures, are outlined in A Manual of Methods for Baculovirus Vectorsand Insect Cell Culture Procedures.

The modified hepatitis E virus capsid protein of the present inventioncan be cloned into the baculovirus vector, and used to infectappropriate host cells (see, for example, O'Reilly et al., “BaculovirusExpression Vectors: A Lab Manual,” Freeman & Co. 1992.). An insect cellline (e.g., Sf9 or Tn5) can be transformed with a transfer vectorcontaining polynucleic acids which encodes the modified HEV capsidproteins of the invention. The transfer vector includes, for example,linearized baculovirus DNA and a plasmid containing the desiredpolynucleotides. The host cell line may be co-transfected with thelinearized baculovirus DNA and a plasmid in order to make a recombinantbaculovirus.

Purification of the virus-like particles of the present invention can becarried out according to the standard technique in the art (See, Li T C,et al., J Virol. 1997 October; 71(10):7207-13. Li T C, et al., J Virol.2005 October; 79(20):12999-3006. Niikura M et al, Virology 2002; 293:273-280). The purified VLPs are then resuspended in a suitable buffer.

VI. Encapsulation Of Heterologous Nucleic Acids

Another aspect of the invention relates to the encapsulation of aheterologous nucleic acid in HEV virus-like particles (See, DNAvaccine-encapsulated virus-like particles derived from an orallytransmissible virus stimulate mucosal and systemic immune responses byoral administration, Gene Therapy 2004. 11, 628-635. S Takamura, MNiikura, T-C Li, N Takeda, S Kusagawa, Y Takebe, T Miyamura and YYasutomi). Any standard technique in the art can be used to encapsulatea heterologous nucleic acid into the VLPs of the present invention. Thegeneral procedure involves (1) disassembling the VLPs formed by themodified HEV capsid protein according to the present invention; and (2)reconstructing the VLPs in the presence of a heterologous nucleic acid.A skilled artisan would recognize that it is preferred to have purifiedVLPs before the encapsulation procedure. It is particularly preferred tohave the VLPs depleted of, or substantially depleted of, any undesirednucleic acids before the encapsulation procedure.

Disassembly of VLPs can be carried out using any standard technique inthe art. Reconstituted virus-like particle can be produced underphysiological conditions (See, US Patent Publication No.: 20080131928).Often, disassembly of virus-like particles require an agent to disruptthe assembly of VLPs, such as a reducing agent or a chelating agent(See, US Patent Publication No.: 20040152181). A skilled artisan wouldrecognize that factors and conditions that affect assembly anddisassembly include: pH, ionic strength, posttranslational modificationsof viral capsid proteins, disulfide bonds, and divalent cation bonding,among others. For example, the importance of cation bonding,specifically calcium, in maintaining virion integrity has been shown forpolyomavirus (Brady et al., J. Virol, 23:717-724, 1977), and rotovirus(Gajardo et al., J. Virol, 71:2211-2216, 1997). Also, disulfide bondsappear to be significant for stabilizing polyomavirus (Walter et al.,Cold Spring Har Symp. Quant. Biol, 39:255-257, 1975; Brady et al., J.Virol, 23:717-724, 1977); and SV40 viruses (Christansen et al., J.Virol, 21:1079-1084, 1977). Also, it is known that factors such as pHand ionic strength influence polyomavirus capsid stability, presumablyby affecting electrostatic interactions (Brady et al., J. Virol,23:717-724, 1977; Salunke et al., Cell, 46:895-904, 1986; Salunke etal., Biophys. J, 56:887-900, 1980). Also, it is known thatpost-translational modifications of some viral capsid proteins mayaffect capsid stability and assembly, e.g., glycosylation,phosphorylation, and acetylation (Garcea et al., Proc. Natl. Acad. Sci.USA, 80:3613-3617, 1983; Xi et al., J. Gen. Virol, 72:2981-2988, 1991).Thus, there are numerous interrelated factors which may affect capsidstability, assembly and disassembly.

Preferably, the VLPs of the present invention is disassembled by theremoval of calcium ions (See, Touze A, Coursaget P. In vitro genetransfer using human papillomavirus-like particles. Nucleic Acids Res1998; 26:1317-1323; Takamura et al., DNA vaccine-encapsulated virus-likeparticles derived from an orally transmissible virus stimulate mucosaland systemic immune responses by oral administration. Gene Therapy 2004;11:628-635). According to the present invention, a reducing agent or achelating agent or both are used to disassemble the VLPs. Variousreducing agents can be used. Preferred embodiments of the reducingagents include, but are not limited to, dithiothreitol (DTT). Variouschelating agents can be used, e.g., ethylene glycol tetraacetic acid(EGTA) or ethylenediaminetetraacetic acid (EDTA). Examples of VLPdisassembly conditions include, but are not limited to, the following:purified VLPs were disrupted by incubation of a buffer containing 50 mMTris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA and 20 mM dithiothreitol for30 minutes.

A skilled artisan would also recognize that complete disassembly of theVLPs is not required, although preferred, to encapsulate a heterologousnucleic acid. An artisan would also recognize that, on other occasions,it is preferred to have partial disassembly of the VLPs. According tothe present invention, the conditions for the partial disassembly of theVLPs can be controlled to still allow efficient encapsulation of aheterologous nucleic acid. Partial disassembly of the VLPs can beachieved by treatment of VLPs with reducing agents alone (e.g., 20 mMDTT) (Sapp et al, J. Gen. Virol., 76:2407-2412, 1995.). According to thepresent invention, once the VLPs are disassembled completely orpartially, encapsulation of a heterologous nucleic acid can be carriedout by reassembling the VLPs in the presence of a heterologous nucleicacid.

In some embodiments of the present invention, reassembly of the VLPs isachieved by re-supplementation of calcium ions to the disrupted VLPs.Alternatively, reassembly of the VLPs is achieved by removal of thereducing agents or the chelating agents. Optionally, factors such as pHand ionic strength, other factors described in the present invention,can be adjusted to achieve efficient reassembly of the VLPs andefficient encapsulation of the heterologous nucleic acid. Examples ofVLP disassembly conditions include, but are not limited to, thefollowing:

Following 30 min of incubation at room temperature, a heterologousnucleic acid in 50 mM Tris-HCl buffer (pH 7.5) and 150 mM NaCl was addedto the disrupted VLP preparation. The disrupted VLP preparation was thenrefolded by incubation for 1 h with increasing concentrations of CaCl2up to a final concentration of 5 mM. VLPs were pelleted byultracentrifugation and resuspended in 10 mM potassium-MES buffer (pH6.2). At each step, the VLP structure formation was confirmed byelectron microscopy after negative staining, as described previously. Toestimate the amounts of encapsulated plasmid DNA, refolded and purifiedVLPs were treated with 10 IU benzonase (SIGMA-ALDRICH, Irvin, UK) for 1h at 20° C. to remove DNA on the surfaces of VLPs and disrupted withEGTA (1 mM). Absorbance of the supernatant was measured for detection ofplasmid DNA contents.

One aspect of the invention relates to encapsulation of a heterologousnucleic acid with a modified HEV capsid protein, wherein the heterlogousnucleic acid comprises an DNA expression cassette encoding an antigen ofnon-HEV source. According to the present invention, the DNA expressioncassette may comprise a promoter sequence and a terminator sequence.

One advantage of the compositions and methods of the present inventionis that the VLPs of the present invention can carries both peptideantigens and nucleic acid antigens. The peptide antigen is presented tothe immune system in associated with MHC class II molecule. Theencapsulated nucleic acids will be presented in the context of MHC classI systems.

In some embodiments of the invention, the heterologous peptide of thepresent invention (peptide antigen) and the heterologous nucleic acidsof the present invention (nucleic acid antigen) are from the differentsources. In some embodiments of the invention, the heterologous peptideof the present invention (peptide antigen) and the heterologous nucleicacids of the present invention (nucleic acid antigen) present differentantigenic epitopes. In particularly preferred embodiments, theheterologous peptide of the present invention (peptide antigen) and theheterologous nucleic acids of the present invention (nucleic acidantigen) are from the same source, e.g., from the same virus. In mostpreferred embodiments, the heterologous peptide of the present invention(peptide antigen) and the heterologous nucleic acids of the presentinvention (nucleic acid antigen) present the same antigenic epitope orepitopes from a single pathogen. This provides synergistic effects instimulating immune response against the same pathogen, thereforeproviding enhanced protection against a disease caused by the pathogen.

The size and number of the heterologous nucleic acids are controllable.According to the present invention, depending on the numbers of desirednucleic acids antigens to be presented, the amount and types of theheterologous nucleic acids can vary. In some embodiments of theinvention, it is desired to present one epitope using the encapsulatednucleic acid, a number of copies of the heterologous nucleic acids canbe allowed to be encapsulated within the VLPs. In other embodiments ofthe invention, it is desired to present 2 or more epitopes using theencapsulated nucleic acids, fewer copies of the heterologous nucleicacids would then be encapsulated within the VLPs.

The size of the VLPs can vary when different constructs of the modifiedhepatitis E virus capsid protein are used. For example, the N-terminalportion of the modified hepatitis E virus capsid protein can be adjustedto increase or decrease the size and encapsulation capacity of the VLPs.In some embodiments of the invention, in constructing the modifiedhepatitis E virus, a portion of HEV ORF 3 protein fused to theN-terminal of a portion of HEV ORF 2 proteins to adjust the size of theVLPs.

IX. Pharmaceutical Compositions, Formulations And Administration

The present invention also provides pharmaceutical compositions orphysiological compositions comprising a heterologous nucleic acidencapsulated in a chimeric VLP formed by the modified HEV capsid proteinof the present invention. Such pharmaceutical or physiologicalcompositions also include one or more pharmaceutically orphysiologically acceptable excipients or carriers. Pharmaceuticalcompositions of the invention are suitable for use in a variety of drugdelivery systems. Suitable formulations for use in the present inventionare found in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Philadelphia, Pa., 17th ed. (1985). For a brief review ofmethods for drug delivery. See Langer, Science 249: 1527-1533 (1990).

The compositions of the present invention can be administered to a hostwith an excipient. Excipients useful for the present invention include,but are not limited to, vehicles, binders, disintegrants, fillers(diluents), lubricants, glidants (flow enhancers), compression aids,colors, sweeteners, preservatives, suspending/dispersing agents, filmformers/coatings, flavors and printing inks

Various adjuvants can be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art. Further adjuvants that can beadministered with the compositions of the invention include, but are notlimited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS 21,QS18, CRL1005, Aluminum salts, MF 59, and Virosomal adjuvant technology.The adjuvants can also comprise a mixture of these substances.

One advantage of the compositions and methods of the present inventionis that the compositions of the present invention can be administered tostimulate immune response without an adjuvant. Therefore, thecompositions of the present invention are in some cases administered toa host without an adjuvant.

Another advantage of the present invention is that the compositions ofthe present invention are suitable for oral delivery. Because mucosalsurfaces are inter-connected, stimulation of one mucosal surface by anantigen can induce mucosal immunity not only on the directly stimulatedsurface, but also on the distant ones. For example, oral delivery of thecompositions of the present invention can protect against respiratoryand genital-urinary infections. The compositions of the presentinvention are also suitable for mucosal delivery, such as delivery tothe buccal or labial mucosa or the respiratory tract mucosa, includingthe nasal mucosa.

The pharmaceutical compositions of the present invention can beadministered by various routes, e.g., oral, subcutaneous, transdermal,intramuscular, intravenous, or intraperitoneal. The preferred routes ofadministering the pharmaceutical compositions are oral delivery at dailydoses of about 0.01-5000 mg, preferably 5-500 mg. The appropriate dosemay be administered in a single daily dose or as divided doses presentedat appropriate intervals, for example as two, three, four, or moresubdoses per day.

For preparing pharmaceutical compositions of the present invention,inert and pharmaceutically acceptable carriers are used. Thepharmaceutical carrier can be either solid or liquid. Solid formpreparations include, for example, powders, tablets, dispersiblegranules, capsules, cachets, and suppositories. A solid carrier can beone or more substances that can also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, or tabletdisintegrating agents; it can also be an encapsulating material.

In powders, the carrier is generally a finely divided solid that is in amixture with the finely divided active component, e.g., a chimericvirus-like particles with an encapsulated nucleic acid. In tablets, theactive ingredient (a chimeric virus-like particles with an encapsulatednucleic acid) is mixed with the carrier having the necessary bindingproperties in suitable proportions and compacted in the shape and sizedesired.

For preparing pharmaceutical compositions in the form of suppositories,a low-melting wax such as a mixture of fatty acid glycerides and cocoabutter is first melted and the active ingredient is dispersed thereinby, for example, stirring. The molten homogeneous mixture is then pouredinto convenient-sized molds and allowed to cool and solidify.

Powders and tablets preferably contain between about 5% to about 70% byweight of the active ingredient. Suitable carriers include, for example,magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin,dextrin, starch, tragacanth, methyl cellulose, sodium carboxymethylcellulose, a low-melting wax, cocoa butter, and the like.

The pharmaceutical compositions can include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component (with or without other carriers)is surrounded by the carrier, such that the carrier is thus inassociation with the compound. In a similar manner, cachets can also beincluded. Tablets, powders, cachets, and capsules can be used as soliddosage forms suitable for oral administration.

Liquid pharmaceutical compositions include, for example, solutionssuitable for oral or parenteral administration, suspensions, andemulsions suitable for oral administration. Sterile water solutions ofthe active component (e.g., a chimeric virus-like particles with anencapsulated nucleic acid) or sterile solutions of the active componentin solvents comprising water, buffered water, saline, PBS, ethanol, orpropylene glycol are examples of liquid compositions suitable forparenteral administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, detergents, and the like.

Sterile solutions can be prepared by suspending the active component(e.g., a chimeric virus-like particles with an encapsulated nucleicacid) in the desired solvent system, and then passing the resultingsolution through a membrane filter to sterilize it or, alternatively, bydissolving the sterile compound in a previously sterilized solvent understerile conditions. The resulting aqueous solutions may be packaged foruse as is, or lyophilized, the lyophilized preparation being combinedwith a sterile aqueous carrier prior to administration. The pH of thepreparations typically will be between 3 and 9, more preferably from 5to 8, and most preferably from 6 to 7.

The pharmaceutical compositions of the present invention can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from a condition in an amount sufficient to prevent,cure, reverse, or at least partially slow or arrest the symptoms of thecondition and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use will depend on the severity of the disease or condition and theweight and general state of the patient, but generally range from about0.1 mg to about 2,000 mg of the composition per day for a 70 kg patient,with dosages of from about 5 mg to about 500 mg of the composition perday for a 70 kg patient being more commonly used.

In prophylactic applications, pharmaceutical compositions of the presentinvention are administered to a patient susceptible to or otherwise atrisk of developing a disease or condition, in an amount sufficient todelay or prevent the onset of the symptoms. Such an amount is defined tobe a “prophylactically effective dose.” In this use, the precise amountsof the composition again depend on the patient's state of health andweight, but generally range from about 0.1 mg to about 2,000 mg of theinhibitor for a 70 kg patient per day, more commonly from about 5 mg toabout 500 mg for a 70 kg patient per day.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of composition of the present invention sufficient toeffectively stimulate immune response in the patient, eithertherapeutically or prophylatically.

XI. EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Examlpe 1 The Capsid Structures of Hepatitis E Virus (HEV)

Hepatitis E accounts for the majority of enterically transmitted non-A,non-B hepatitis worldwide. In countries with poor sanitary conditionsand high population density, hepatitis E causes water-borne epidemicswith substantial mortality rates (20%) in pregnant women (Zhou et al.,2005. Vaccine 23:3157-65.). The causative agent is the hepatitis E virus(HEV), a non-enveloped single-stranded positive-sense RNA virus,belonging to the Hepevirus genus in the Hepeviridae family (Emerson etal., 2004. Hepevirus. Elsevier/Academic Press, London.).

The HEV virion has a diameter about 32 to 34 nm and encapsidates a 7.2kb genome. The full-length genome contains three open reading frames(ORFs), one of which, ORF2, maps to the 3′ terminus and encodes the 660amino acids viral capsid protein. Like other hepatitis viruses, HEV isunable to propagate in large quantities in current cell culture systems.Preventive strategies rely on recombinant protein vaccines derived fromthe HEV capsid protein (Maloney et al., 2005. Vaccine 23:1870-4.).

Self assembled virus-like particles (VLPs) from the HEV structuralprotein were observed when a construct of the ORF2 protein lacking theRNA-binding N-terminal 111 amino acids was expressed in the Tn5 insectcell line. These particles, measuring 27 nm in diameter, were muchsmaller than the native virion; but nevertheless, they were recognizedby anti-HEV sera and could induce protective immunity in Cynomolgusmacaques (Li et al., 2004. Vaccine 22:370-7.). Therefore, essentialepitopes are well-preserved and remain exposed on the surface of thesesmall particles, making them a potential viable vaccine (Li et al.,2001. Vaccine 19:3476-84; Maloney et al., 2005. Vaccine 23:1870-4.).Analysis showed that the recombinant HEV VLPs were assembled of the ORF2protein truncated at the C-terminus by a Tn5 cell furin-like activity(Li et al., 2005. J Virol 79:12999-3006; Li et al., 1997. J Virol71:7207-13.). Expression of the HEV ORF2 protein in other cellsdemonstrated that removal of the C-terminal 52 amino acid (aa) residuesis a prerequisite to VLP formation. With this knowledge, severalself-assembling recombinant HEV VLPs have been constructed,demonstrating that the residues 126 to 601 are the essential elementsfor the initiation of VLP assembly (Li et al., 2005. J Virol79:12999-3006.).

Three-dimensional structures of the recombinant VLP expressed in Tn5(VLPTn5) and Sf9 (VLPSf9) cells have been determined by electroncryomicroscopy (cryo-EM) at the resolutions of 22 and 23 Å,respectively. Structural studies revealed that the recombinant HEV VLPis an empty T=1 icosahedral particle containing 30 spike-likeprotrusions extending from the capsid surface (Li et al., 2005. J Virol79:12999-3006; Xing et al., 1999. Virology 265:35-45.). Density analysisrevealed that the truncated ORF2 protein contains two distinct domains;namely, a shell (S) domain, which forms the continuous surface of theviral capsid, and a protrusion (P) domain, which forms the protrudingspikes. Neighboring protein subunits at each icosahedral 2-fold axiscombine to form the 30 protrusions. The internal cavity of therecombinant HEV VLP has a diameter of 9.3 nm, which is insufficient toencapsidate a full length RNA genome; hence, there is no significant RNAdensity found within the particle in the three-dimensionalreconstruction (Xing et al., 1999. Virology 265:35-45.).

Similar studies using the recombinant truncated ORF2 protein (aaresidues 112-607) from Sar55 strain expressed in Sf9 cells failed todetect VLP formation. The monoclonal antibodies (MAbs) raised againstthe truncated ORF2 or isolated by phage display from a chimpanzee cDNAlibrary neutralized the HEV SAR-55 strain in vivo (Schofield et al.,2000. J Virol 74:5548-55.). These antibodies were mapped to recognizethe epitope located at residues 578-607 by radio-immunoprecipitationassays. A further characterization of the antigenic sites of thetruncated ORF2 protein (aa residues 112-607) of the Sar55 strain showedthat the antigenic site located at the C-terminal region overlaps withthe epitope recognized by neutralizing antibodies, and the C-terminus isimportant for enhancing the presentation of this epitope (Schofield etal., 2003. Vaccine 22:257-67.).

The location of the HEV ORF2 C-terminus has not yet been experimentallyidentified. Sequence analysis indicates, however, that this site issolvent accessible. The HEV ORF2 aggregation into VLP is a key factorfor assembly of the conformation-dependent epitopes in a functional form(Maloney et al., 2005. Vaccine 23:1870-4.). Taking into considerationthat the HEV neutralizing antigenic epitope is conformation-dependentand located in the C-terminal region ORF2 proteins (Meng et al., 2001.Virology 288:203-11; Schofield et al., 2003. Vaccine 22:257-67.), wehave conducted a structural analysis of the C-terminal region in thecontext of HEV VLP assembled of the truncated ORF2 protein using cryo-EMand three-dimensional reconstruction techniques. In the course of thestudy we determined the structure of a chimeric recombinant VLPcontaining a B-cell tag of 11 amino acids inserted after residue 608(Niikura et al., 2002. Virology 293:273-80.) and a recombinant VLPassociated with Fab fragments from the mouse monoclonal antibody againstresidues 595-601. Additionally, three-dimensional structure waspredicted for the C-terminal region at position 525 to 608, which ispartially included in the HEV neutralizing epitope (Meng et al., 2001.Virology 288:203-11.). It is important to note that the results fromthree-dimensional structure prediction concur with those from cryo-EM,revealing that the binding footprint at the C-terminal region covers thelateral side of the P domain, including the neutralizing epitope of ORF2protein, and terminates at the spike surface.

Materials and Methods

Production and purification of IgGs: Eight-week-old female BALB/c micewere immunized at 0 and 4 weeks by intraperitoneal inoculation with HEVVLPs (100 ug/ml). Four weeks later, a final boost of equal volume ofantigen was administered. Three days after the final boost, mouse spleencells were fused with P3U1 mouse myeloma cells using polyethylene glycol1500 (50% [wt/vol]) (Boehringer, Mannheim, Germany) as essentiallydescribed by Adler and Faine (Adler et al., 1983. Pathology 15:247-50).Supernatant from microplate wells positive for hybridoma growth wasscreened by enzyme-linked immunosorbent assay (ELISA) using therecombinant HEV VLPs as antigen. Hybridomas secreting specificantibodies to HEV were subcloned three times by limiting dilution, afterwhich they were considered to be monoclonal. Antibodies in thesupernatants were isotyped using the Mouse Monoclonal Antibody Isotypingkit (Amersham, Little Chalfont, Buckinghamshire, U.K.) in accordancewith the manufacturer's protocol. Hybridomas were grown in bulk instationary flasks (Nunc, Roskilde, Denmark) using PRMI-1640 with 15%FCS. Supernatant was harvested and antibodies were purified using HiTrapprotein G affinity columns (Pharmacia Biotech AB, Uppsala, Sweden) andstored at −80 C. The mouse monoclonal antibody MAb 224 hasimmunoglobulin G1 (IgG1) isotype.

Preparation of Fab fragments: A method employing papain cleavage wasused to yield the isolated Fab fragments from purified MAb224. Areducing L-cystein buffer was used to activate papain, and the MAb224was mixed with papain in a 100:1 molar ratio. The cleavage mixture wasincubated overnight at 30 ° C. The reaction was quenched by addingiodacetamide and analyzed on SDS-PAGE. The papain digestion product waspurified using a Protein-A column according to the manufacturer'sinstruction. The Fc fragments and un-cleaved MAbs224 were immobilized onthe column in binding buffer while the Fab fragments were collected inthe flow-through fractions.

Purification of the VLPs: The construct of recombinant baculoviruses wasprepared as described (Li et al., 2005. J Virol 79:12999-3006; Li etal., 1997. J Virol 71:7207-13; Niikura et al., 2002. Virology293:273-80.); as VLP/C-tag, B-cell tag epitope on glycoprotein D ofherpes simplex virus “QPELAPEDPED” (SEQ ID NO:7) was inserted afteramino acid 608. The production and purification of HEV VLPs wereconducted as described (Li et al., 2005. J Virol 79:12999-3006; Li etal., 1997. J Virol 71:7207-13; Niikura et al., 2002. Virology293:273-80; Xing et al., 1999. Virology 265:35-45.). Briefly, the DNAfragments containing the N-truncated ORF2 protein (for VLPTn5),N-and-C-truncated ORF2 protein (for VLPSf9) andN-truncated-and-C-inserted ORF2 protein (contains VLP/C-tag) were clonedwith baculovirus transfer vector pVL1393 to yield pVLORF2. Insect Sf9cells (Riken Cell Bank, Tsukuba, Japan) were co-transfected with pVLORF2and the linearized wild-type Autographa californica nuclear polyhedrosisvirus DNA (Pharmingen BaculoGold™ #21100D) by the lipofectin-mediatedmethod to produce recombinant baculoviruses. The recombinant baculoviruswas plaque-purified three times. Both Sf9 and Tn5 cells, the latter froma Trichoplusia ni, BTL-Tn 5B1-4 (Tn5) (Invitrogen, San Diego, Calif.)(Wickham et al., 1993) were infected with the recombinant baculovirusesat a m.o.i.>5 and incubated in EX-CELL™ 405 medium (JRH Biosciences,Lenexa, Kans.) for 6 days at 26.5° C. The supernatant was collected andintact cells, cell debris and progeny baculoviruses were removed bycentrifugation at 10,000g for 90 min. The supernatant was then spun at30,000 rpm for 2 h in a Beckman SW32 Ti rotor. The resulting pellet wasresuspended in 4.5 ml EX-CELL™ 405 and stored at 4° C. After mixing with1.96 g of CsCl, the sample was centrifuged at 35,000 rpm for 24 h at 4°C. in a Beckman SW55Ti rotor. The white band was harvested by puncturingthe tubes with a 22-gauge needle, diluted 4 times with EX-CELL™ 405 andcentrifuged for 2 h in a Beckman TLA 55 rotor at 45,000 rpm to removeCsCl. The VLPs were re-suspended in 100-500 μl of EX-CELL™ 405 andstored at 4° C. The material was later diluted with 10 mM potassium-MESbuffer.

Preparation of VLP-Fab complexes for cryo-electron microscopy: TheVLP/Fab224 complexes were prepared by incubating Fab224 fragments withVLP purified from Sf9 cells (VLPsf9) at a molar ratio exceeding 1:300 in10 mM pH 6.2 potassium-MES buffer at 4° C. over night. High purityVLP/Fab224 complexes were obtained using a short column containingSephacryl-300 to remove the unbound Fab fragments from the sample inorder to reduce the background densities for the subsequent structuraldetermination. The fractions containing VLP/Fab224 complexes werecollected based on reading UV spectro-photometer at an optical density(OD) of 280 nm. The Fab binding occupancy was roughly estimated bySDS-PAGE, in which the purified VLP/Fab224 complexes were loaded on anacrylamide gel (gradient 8-25%) and electrophoresis was run on a Phast™system (Pharmacia) under constant-voltage condition. The integrity ofparticles was checked by negative stained electron microscopy (EM) using2% uranyl acetate (UA).

Cryo-electron microscopy: The sample preparation and cryo-EM operationwere followed well established procedures described previously (Li etal., 2005. J Virol 79:12999-3006; Xing et al., 1999. Virology265:35-45.). Briefly, a drop containing 3.5 μl of sample was applied ona glow-discharged homemade holey-carbon grid, blotted with a piece offilter paper for 3 s to remove the extra liquid and quickly plunged intoliquid ethane cooled by liquid nitrogen in a homemade cryo-container.Samples were frozen in a thin layer of vitrified ice. The grid was thentransferred into a Gatan 626DH (Gatan Inc, CA) cryo-holder and kept atthe low temperature environment (−178 ° C.) for the subsequentprocessing. Micrographs were recorded under low-dose condition (<10e-/Å2) using Kodak 50163 films at a magnification of 45,000× on anPhilips CM-120 electron microscopy operated at 120 kV and photographedat the defocus range from 1000 to 3000 nm. Micrographs were visuallyinspected and selected based on the criteria of suitable particleconcentration, optimal ice thickness and minimal specimen drift. Onlythose micrographs fulfilling these criteria were analyzed.

Image processing: Selected micrographs were digitized using a HeidelbergPrimescan D8200 (Heidelberg, Germany) at a 14 μm scanning step size,corresponding to 3.11 Å per pixel at specimen space. Particles weremanually picked using Robem (v2.14), and centered by cross-correlatingeach of them against the circular average image. The astigmatism and thedefocus value were evaluated by the average sum of the power spectrafrom all particles within single micrograph. The first zero of the dataused for the following structural determination was approximately withinthe range of 17-20 . The self-common line algorithmic was used to yieldthe initial models for VLP/C-tag and VLP/MAb224 (Crowther, 1971. PhilosTrans R Soc Lond B Biol Sci 261:221-30.), respectively. The followingorigins and orientations search of each particle was carried outiteratively using polar Fourier transformation (PFT) algorithm(pftsearch program) running on an AMD MP1800 MHz dual-processors Linuxworkstation (Baker & Cheng. 1996. J Struct Biol 116:120-30.).Three-dimensional reconstructions were computed by combining a set ofparticles of orientations spreading evenly in an icosahedral asymmetricunit, with the Fourier-Bessel algorithm and superimposing of 5-3-2icosahedral symmetry (em3dr program). To examine the reliability of thethree-dimensional reconstruction, at the final refinement step, thedataset was evenly divided into two parts and computed twothree-dimensional reconstructions, respectively. The resolution wasestimated using Fourier shell correlation (FSC) by assessing theagreement between these two reconstructions in the Fourier space; acoefficient value of 0.5 was used as the criteria, the estimatedresolutions of the three-dimensional reconstructions of VLP/C-tag andVLP/Fab224 were computed to 17.5 Å and 18.5 Å, respectively (FIG. 2C).

The three-dimensional reconstructions were rendered and visualized usingRobem and PyMOL (DeLano, 2002. The PyMOL Molecular Graphics System.DeLano Scientific, Palo Alto, Calif., USA.). The contour level waschosen at the value corresponding to the 100% mass of the particlevolume. The electron density map was inspected in the isosurface mode,which builds a surface barrier to contour the density about a certainthreshold, provided the concise surface details on the density map.

Difference maps analysis: Difference maps were computed by firstsearching the magnification factor between two three-dimensional modelsand adjusted them to have the minimum difference using cmpEM. As thesearching results showed all the models having the size difference lessthan 0.5%, the magnification factor was set to 1. Then, all thedensities corresponding to the S domain were radially scaled bymultiplied a fixed factor and added a constant using cmpEM. Thedifference maps were obtained by subtracting VLPTn5 from VLP/C-tag andVLPSf9 from VLP/Fab224. The models of VLPTn5 and VLPSf9 were taken frompreviously study (Li et al., 2005. J Virol 79:12999-3006.). Theresulting difference maps were set the solvent density to zero at theradii smaller than inner surface of S domain and the radii beyond thelargest radii of VLP/C-tag and VLP/Fab224. The contour level were chosento match the mass of tag using an average protein density of 1.36 g/cm3and the estimated molecule weight of Fab224 was used 45 kDa.

Structural prediction on partial truncated HEV ORF2 protein: Thestructure prediction was carried out by two steps methods; namely (A)Domain parsing and (B) De novo three-dimensional structure prediction.

(A) Domain parsing: to predict the three-dimensional model of theC-terminal region of the truncated ORF2 protein (residues 112-608), adomain prediction method Ginzu (Kim et al., 2005. Proteins 61 Suppl7:193-200.) was used as the first step for structure prediction. Ginzuis a sequential procedure for detection of putative domains. It firstperforms homologous structure searches to detect regions in a querysequence that are homologous to experimentally determinedthree-dimensional structures. BLAST, PSIBLAST (Altschul et al., 1997.Nucleic Acids Res 25:3389-402.), FFAS03 (Jaroszewski et al., 2002.Protein Sci 11:1702-13; Rychlewski et al., 2000. Protein Sci 9:232-41.),and 3D-Jury (Ginalski et al., 2003. Bioinformatics 19:1015-8; Ginalski &Rychlewski, 2003. Nucleic Acids Res 31:3291-2.) are used for this step.In the case of regions without homologous structures, Ginzu continueswith a search against Pfam-A using HMMER (Cheng et al., 1994. Structure2:271-82.) and then parsing by multiple sequence alignment (MSA) basedmethods to predict putative domains. Since the truncated ORF2 proteinsequence did not match any homologous structure, selecting cut pointsbetween the domains was done using an MSA of the full-length ORF2protein target derived from PSI-BLAST search against NCBI non-redundant(NR) protein sequence database. The most populated non-overlappingclusters of sequences in the MSA were assigned as domains, and the finalcut points were made at positions that have high incidence of sequencetermini, a strong loop prediction (as determined by PSIPRED (Jones,1999. J Mol Biol 292:195-202.)), and reduced occupancy of alignedresidues. Boundaries were assigned so that the putative domains remainedwithin size limits (-200 residues) approachable to the de novothree-dimensional structure prediction protocol of the Rosetta softwarepackage (Bonneau et al., 2001. Proteins 43:1-11; Bonneau et al., 2001.Proteins Suppl 5:119-26.). Hence, the truncated ORF2 protein was brokenup into putative five domains and the domain corresponding to theC-terminal region contains 84 amino acids (residues 525-608).

(B) De novo three-dimensional structure prediction: thethree-dimensional structure of this domain was modeled using the Rosettade novo protocol, as implemented in the Rosetta server (Kim et al.,2004. Nucleic Acids Res 32:W526-31.). For each putative domain, three-to nine-residue fragment libraries representing local conformationspresent in the protein database (PDB) were generated and then assembledinto models by fragment insertion using a scoring function that favorsprotein-like features (Simons et al., 1997. J Mol Biol 268:209-25;Simons et al., 1999. Proteins 34:82-95.). Ten thousand decoys for theoriginal C-terminal region of the truncated HEV ORF2 query protein and5000 decoys for up to two sequence homologs were generated. From thisset of decoys, 2000 query decoys and 1000 decoys from the sequencehomologs were selected based on score and filtering out decoys withunfavorable number of local contacts or strand topologies. The selecteddecoys were then clustered based on Ca root-mean-square deviation (RMSD)over all ungapped positions. The top 9 cluster centers were chosen asthe best ranked models, and the best scoring model that passed all ofthe above mentioned filters was selected as the 10th model. The modelshaving the best 10 ranking scores were then used for fitting intocryo-EM density maps.

Fitting the X-ray Atomic model of the truncated ORF2 protein intocryo-EM density maps of chimeric HEV VLP and VLP/Fab224: Manuallyfitting was carried out by translational and rotational movement of thepredicted three-dimensional atomic models into the cryo-EM density mapsusing program O (Jones et al., 1991. Acta Crystallogr A 47 (Pt2):110-9.). The contour level of cryo-EM density maps was chosen toensure the volume rendered at 100% mass assuming a protein density of1.36 g/cm3. To achieve the best fit of the models in cryo-EM densitymaps, the entire atomic model was treated as a rigid body; the fittingprocedure used the criteria that the residues 595-601 need to be exposedat the surface as well as located closed to the density of MAb224-Fab;in the meantime, the C-terminus of the predicted model should also pointtoward to the surface of the particle. To optimize the fitting results,symmetry-related molecules were generated and judged by the crashesbetween each molecule. Only one out of the best ten models was found tomeet the fitting criteria by visual inspection. The figures wereprepared using the program PyMOL (DeLano, 2002. The PyMOL MolecularGraphics System. DeLano Scientific, Palo Alto, Calif., USA.).

Results

MAb224 recognizes residues 595-601 of the HEV ORF2 protein: A Westernblotting assay was performed to characterize the epitope recognized byantibody MAb224. A series of C-terminally truncated ORF2 proteins wereseparated using 10% SDS-PAGE, and blotted with MAb224 (FIG. 1A).Truncated ORF2 proteins comprising residues 112-600, 112-596, and112-589 were unable to interact with MAb224. These data indicate thatresidue 601 is the most important for MAb224 binding. Taking intoconsideration that antigenic epitopes are usually short regions of 3-7aa long, Mab224 binding site should locate within aa position 595-601 ofthe ORF2 protein and it is within the neutralization epitope localizedat residues 578-607 reported by Schofield et al (Schofield et al., 2000.J Virol 74:5548-55.). The lower molecular weight protein detected withMAb224 are products of degraded ORF2 protein at the N-termini.

The C-terminal markers: We used two markers for the identification ofposition of the C-terminal region in three-dimensional structure asshown in FIG. 1B. First, a chimeric recombinant HEV VLP containing the11 aa B-cell tag epitope inserted at aa 608 (VLP/C-tag) was used to mapthis epitope in VLP. Results from both immunoprecipitation andenzyme-linked immunosorbent assay (ELISA) experiments suggest that thetag epitope is exposed on the particle surface (Niikura et al., 2002.Virology 293:273-80.). Second, MAb224 epitope mapped at 601-608 was usedto detect this region in VLP particles. It was found that MAb224 reactedwith the intact recombinant HEV VLP in an ELISA experiment, suggestingthat the region at position 601-609 is exposed on the BLP surface. Toincrease the density of the MAb224 binding to VLP; we used Fab fragmentsinstead of MAb224 in the IgG form. For this purpose, MAb224 was treatedwith papain and the Fab fragments were purified. The resulting Fabfragments were incubated with VLP overnight to obtain VLP-Fab complexes(VLP/MAb224) for the subsequent data collection.

Two-dimensional electron cryo-micrographs: Cryo-EM specimens ofVLP/C-tag and VLP/MAb224 were prepared by standard procedures (Li etal., 2005. J Virol 79:12999-3006; Li et al., 1997. J Virol 71:7207-13.).The samples were imaged at a magnification of 45,000× in a PhilipsCM-120 EM operated at 120 kV. Digitized cryo-electron micrographs offrozen-hydrated samples embedded in the thin layer of vitreous ice shownthat both particles had circular profiles with spiky densities extendingfrom the surface (FIG. 1C). Both particles appeared as empty cores,suggesting the absence of RNA moiety, which is completely consistentwith our previous observation (Li et al., 2005. J Virol 79:12999-3006;Xing et al., 1999. Virology 265:35-45.). The sizes of both particleswere approximately 27 nm without taking into account the extra densitiesextending further from the surface of VLP/MAb224 (FIG. 1C, right).

Three-dimensional reconstruction of VLP/C-tag and VLP/MAb224: A total of782 particles were used to reconstruct the final three-dimensional modelof VLP/C-tag and 615 particles for VLP/MAb224. Surface representationsof cryo-EM structures of VLP/C-tag and VLP/MAb224 exhibited similarfeatures as they both displayed T=1 icosahedral symmetry with60-protein-subunits arranged into 30 dimeric protruding spikes locatedat each icosahedral two-fold axis. In agreement with the previouslypublished cryo-EM structure of VLP (Li et al., 2005. J Virol79:12999-3006.), the surfaces of VLP/C-tag and VLP/MAb224 can also bedivided into two distinct domains, a surface (S) domain and a protrusion(P) domain. The S domain forms a continuous basal shell of the capsidwith small hollows at each icosahedral five-fold axis. The P domainprojects outward from the surface with adjacent protein subunits locatedat two-fold axis forming dimeric arch-like spike. The distance betweeneach two-fold axis at the top surface of P domain was ˜76 Å and that isalso consistent with the results measured in VLPTn5 and VLPSf9 (Li etal., 2005. J Virol 79:12999-3006.). No significant RNA density was foundwithin both structures.

In VLP/C-tag, the particle had a diameter of approximately 280 Åmeasured from three-dimensional reconstruction (FIG. 2A). The P domainsshowed two discernible prominences at the top, which were not observedin VLP/MAb224 (FIG. 2B) or other VLP structures published earlier (Li etal., 2005. J Virol 79:12999-3006.). The prominence has the height about6 Å measured from the top of the P domain. In contrast to structure ofVLP/C-tag, in VLP/MAb224 the diameter of the particle itself without Fabfragments was approximately 270 A; in addition, sixty Fab fragments wereobserved around the particle. MAb224 bound to the shoulder of the Pdomain tilting away from the parallel direction of the two-fold axis.The height of the Fab molecule was measured ˜57 Å as by the long axisperpendicular to the surface of P domain, and along the top view oftwo-fold axis, the adjacent MAb224 has a distance of 95 Å. BothVLP/C-tag and VLP/MAb224 presented here were the density maps renderedat the contour of 100% mass-volume estimated by using an average proteindensity of 1.36 g/cm3. No steric hindrance was observed in the 3Dreconstruction between each Fab molecule around the regions of eitherfive-fold or three-fold axes.

Determining the C-terminal tail on recombinant HEV VLP: Significantdifferences were noted on the top surface of P domain of VLP/C-tagcompared to unmodified VLP. To analyze whether the difference was causedby the inserted tag, a complementary analysis was used to calculate thedifference map by subtracting VLPTn5 from VLP/C-tag. The S domain radiiwere matched, which allowed for analyzing differences between the radiicorresponding to the P domain since the tag was found to be exposed tothe surface. Taking into consideration that the average protein densityis 1.36 g/cm3, the tag volume was estimated to be 1.70 nm3, Theresulting difference map superimposed on VLPTn5 revealed the density ofthe tag located at the top surface of the P domain where the prominenceswere found (FIG. 3A). Similar analysis was applied to VLP/MAb224; thedifference map was calculated by subtracting VLPSf9 from VLP/MAb224revealing the Fab density located at the lateral side of P domain (FIG.3B). The footprint of MAb224 binding covered the residues of 601-608 ofthe C-terminal region according to the western blotting analysis.

Structural prediction for the C-terminal region: The structure of theC-terminal region at position 525-608 was modeled using Rosettaprotocols (Kim et al., 2004. Nucleic Acids Res 32:W526-31.). The modelwith the top 10 scores were used and evaluated by manually fitting eachof them into VLP/C-tag and VLP/MAb224 density maps using the software O(Jones et al., 1991. Acta Crystallogr A 47 (Pt 2):110-9.). As the tagepitope was shown to be exposed on the surface of VLP/C-tag (Niikura etal., 2002. Virology 293:273-80.), those models with tag buried either inthe center of the P domain or under the S domain of VLP were discardedbecause the region 601-608 should be located close to the footprint ofthe MAb224 binding sites. Only one model met the criteria describedabove (FIG. 4). This model contains three alpha-helixes (residues546-550, 591-601 and 604-607), and four beta-strands (residues 526-530,535-540, 563-573, and 577-585). The structure fitting experiment showedthat the C-terminus was located underneath the prominent density in theVLP/C-tag density map (FIG. 5A and 5B) and the alpha-helix (residues604-607) and beta-sheet (residues 577-585) were exposed to the interfacebetween Fab224 and VLP so that the beta-sheet oriented downward from thesurface and connected by a loop curved in a U-shape to an alpha-helixturning upward to the top surface again. The residues 591-607 recognizedby the neutralizing antibody reported by Schofield et al (Schofield etal., 2003. Vaccine 22:257-67.) were also covered by the footprint ofMAb224 (FIG. 6A). As indicated in FIG. 6B, the essential epitope locatednot only at the lateral sides of the capsid protrusions, but also in thevicinity on the top of the protruding surface extended by the C-terminusof the capsid protein.

Discussion

Expressing the truncated HEV ORF2 protein in either Tn5 or Sf9 cellsresults in self-assembled VLPs of similar structure (Li et al., 2005. JVirol 79:12999-3006.). The recombinant VLP and native virion shareantigenic properties because both capable of inducing systemic andmucosal immune responses (Li et al., 2001. Vaccine 19:3476-84.) as wellas the HEV specific neutralizing immune responses (Li et al., 2004.Vaccine 22:370-7.) in experimental animals. Earlier identified cryo-EMthree-dimensional structures showed that the recombinant HEV VLPs form aT=1 icosahedral particle with 30 dimeric protrusions projected ˜43 Åradially above a capsid shell (Li et al., 2005. J Virol 79:12999-3006;Xing et al., 1999. Virology 265:35-45.). As the protrusions are the mostexposed part of the particle, they were suspected to form the antigenicdeterminates. Our results provide the first evidence relating the HEVORF2 protein sequence to its spatial configuration.

Two strategies were independently used for localizing the C-terminus ofthe truncated ORF2 protein in VLP; namely, the eleven-residue peptideC-tag and MAb224 recognizing the C-terminal region at position 601-608.A comparative analysis of the VLPs with and without the C-tag revealed asignificant density difference at the top surface of the P domain aroundthe two-fold axis (FIG. 3A). The shell domain and symmetry appeared tobe identical, suggesting that the inserted tag epitope was not directlyinvolved in the protein-protein interactions during the VLP formation.Superimposition of the difference map on the VLP without tag showed thatthe tag is exposed on the top surface of the P domain (FIG. 3A). Thisfinding is in agreement with a report that the tag epitope is exposed onthe VLP surface since it can be recognized with antibodies against thistag epitope as detected in immuno-precipitation and ELISA experiments(Niikura et al., 2002. Virology 293:273-80.). Additionally, this findingsuggests the use of the recombinant HEV VLP for constructing amultivalent vaccine since the HEV ORF2 C-terminal region not onlycontains the HEV neutralizing epitope but also can successfully presentforeign epitopes such as the C-tag to immunocompetent cells.

The monoclonal antibody (MAb224) can recognize the terminal region atposition 601-608 at shown by the Western blot analysis (FIG. 1A). TheHEV ORF2 proteins truncated at position 600 could not bind to MAb224.This finding suggests that the antigenic site recognized by MAb224 is atleast a part of a linear epitope located at the C-terminus. However, itis conceivable that a conformational epitope at the C-terminus may bepreserved in denaturing condition if the ORF2 protein were to form arigid secondary structure capable of withstanding this condition.Similar results have been obtained by Schofield et al. (Schofield etal., 2000. J Virol 74:5548-55.), who originally hypothesized that theneutralizing epitope located at the C-terminus of the ORF2 protein fromgenotype 1 HEV was linear; but later after examining a shorter peptidecomprising residues 589-607 that failed to interact with theneutralizing antibodies in the denaturing RIPA, suggested that thisepitope(s) is conformational (Schofield et al., 2003. Vaccine22:257-67.). Since the site recognized by MAb224 overlaps with theneutralizing epitope (Li et al., 2005. J Biol Chem 280:3400-6; Meng etal., 2001. Virology 288:203-11; Schofield et al., 2003. Vaccine22:257-67.), the MAb224 binding to the recombinant VLP may be consideredas a model for the neutralizing interaction. We used the Fab fragment(Fab224) produced from Mab224 to study antibody binding to theC-terminal region of the HEV ORF2. This experiment suggests that the HEVneutralization mechanism involves the abrogation of the virus attachmentto cell. In the detailed analysis of the antigenic site located atC-terminus, Schofield et al. showed that the antibodies, HEV#4 andHEV#31, recognized amino acids between 597 and 607 are capable toneutralize HEV (Schofield et al., 2003. Vaccine 22:257-67.), and theepitope recognized by those antibodies is overlapped with the siterecognized by MAb224. The neutralizing activity of MAb224 has not yetbeen examined. However, our three-dimensional reconstruction ofVLP/MAb224 suggests the MAb224 antigenic site is exposed at the lateralsides of the P domain. The long dimension of the MAb224 density extends˜53 Å radially away from the surface of the P domain (FIG. 3B). Thisdata suggests that the Fab224 binding can cause a steric interferencewith the cell receptor recognition. Because the binding footprint ofMAb224 also covers the epitope recognized by HEV#4 and HEV#31 (FIG. 6A),the neutralizing mechanisms of antibodies HEV#4 and HEV#31 might besimilar to Fab224 through blocking the viral attachment pathway.

It has been shown that at least one immunodominant neutralizationepitope is commonly shared by all four major HEV genotypes and thepeptide encompassing the HEV ORF2 region at position 458 to 607 is ableto present this conformational neutralization epitope (Emerson et al.,2006. J Gen Virol 87:697-704; Zhou et al., 2005. Vaccine 23:3157-65.).HEV has only one known serotype. Therefore, this neutralizing epitopeshould be considered as a major component for the development of auniversal vaccine against HEV. The HEV ORF2 protein used in this studyshares 93-94% sequence similarity with this protein from other threegenotypes. Additionally, the spatial configuration of the P domain isconserved in recombinant VLPs for all HEV genotypes (will be publishedelsewhere). Since the P domain contains the neutralizing epitope, thedimeric nature of this domain may present the key structural property ofthis epitope.

We previously reported that the HEV ORF2 protein region 125-601 aa isthe essential elements for VLP assembly. Truncation of the C-terminalregion at position 600 prevents the VLP formation. Li et al. (Li et al.,2005. J Biol Chem 280:3400-6.) hypothesized that the hydrophobic regionat amino acids 597-602 is a site for dimeric interactions; however,neither our cryo-EM 3D structures nor the predicted model of theC-terminal region supports this hypothesis. Our results showed that thepeptide regions (residue 597-602) of the neighboring ORF2 proteinmolecules are distant from each other. Therefore, the possible reasonfor the abrogation of VLP formation may be duo to the decrease instability in the secondary structural element at the C-terminal regionwhich leads to the destruction of dimeric interactions.

In conclusion, the C-terminal region of the HEV ORF2 protein isimportant for both the presentation of the neutralizing epitope andcapsid assembly. In the present study we found the C-terminal regionextends from lateral sides to the top surface of the P domain. Thesignificant surface exposure of this domain is consistent with locationof the major HEV neutralizing epitope in this domain. The structuralanalysis of VLP/C-tag suggests that the HEV ORF2 protein C-terminalregion may be used for insertion of foreign antigenic epitope and,therefore, may serve as a novel platform for engineering multivalentvaccines. A complex structure of MAb224 conjugated to VLP provides adirect evidence that the MAb224 antigenic site is located at the upperside of the P domain. This implies that the region 609-660 removed fromthe recombinant VLP does not obstruct the presentation of thisneutralizing epitope by native virion, and that the P domain observed inthe recombinant VLP is present on the surface of native HEV.

Example 2 Biological and Immunological Characteristics of Hepatitis EVirus-Like Particles Based on the Crystal Structure

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. Thecrystal structure of HEV-like particles (HEV-LP) consisting of capsidprotein was determined at 3.5-Å resolution. The capsid protein exhibiteda quite different folding at the protruding and middle domains from themembers of the families of Caliciviridae and Tombusviridae, while theshell domain shared the common folding. Tyr-288 at the 5-fold axis playskey roles in the assembly of HEV-LP, and aromatic amino acid residuesare well conserved among the structurally related viruses. Mutationalanalyses indicated that the protruding domain is involved in the bindingto the cells susceptive to HEV infection and has some neutralizationepitopes. These structural and biological findings are important forunderstanding the molecular mechanisms of assembly and entry of HEV andalso provide clues in the development of preventive and prophylacticmeasures for hepatitis E.

Hepatitis E is an acute viral hepatitis caused by infection withhepatitis E virus (HEV) that is transmitted primarily by a fecal-oralroute (Panda S K, Thakral D, Rehman S, Rev Med Virol, 17:151-180 (2007);Purcell R H, Emerson S U, J Hepatol, 48:494-503 (2008)). Numerousepidemic and sporadic cases have occurred in developing countries ofAsia, the Middle East, and North Africa, where sanitary conditions arenot well-maintained. Hepatitis E affects predominantly young adults, andHEV infection in pregnancy is one of the risk factors for severe diseaseand death (Navaneethan U, Al Mohajer M, Shata M T, Liver Int,28:1190-1199 (2008)). Recent epidemiological studies show thatsignificant prevalence of HEV and anti-HEV antibody is found in humansand several animals worldwide, even in developed countries (Meng X J, etal., Proc Natl Acad Sci USA, 94:9860-9865 (1997); Sonoda H, et al., JClin Microbiol, 42:5371-5374 (2004); Okamoto H, Virus Res, 127:216-228(2007); Li T C, et al., Emerg Infect Dis, 11:1958-1960 (2005); Yazaki Y,et al., J Gen Virol, 84:2351-2357 (2003)).

HEV is the sole member of the genus Hepevirus within the familyHepeviridae and has a 7.2-kb positive-sense RNA genome (Tam AW, et al.Virology, 185:120-131 (1991)). Five major genotypes have been identifiedso far (Purcell R H, Emerson S U, J Hepatol, 48:494-503 (2008)). Theviruses in the genotypes 1 and 2 are maintained among only humans, whilethose in the genotypes 3 and 4 are found in pigs or wild animals (Meng XJ, et al., Proc Natl Acad Sci USA, 94:9860-9865 (1997); Sonoda H, etal., J Clin Microbiol, 42:5371-5374 (2004); Okamoto H, Virus Res,127:216-228 (2007)). However, infections of human with genotypes 3 and 4via zoonotic transmission or blood transfusion were reported in thedeveloped countries, such as Japan and the United States (Li T C, etal., Emerg Infect Dis, 11:1958-1960 (2005); Yazaki Y, et al., J GenVirol, 84:2351-2357 (2003); Matsubayashi K, et al., Transfusion,44:934-940 (2004)), suggesting that hepatitis E caused by infection withgenotypes 3 and 4 of HEV is an important emerging infectious disease.The viruses in the genotype 5 are of avian origin and are thought to beuninfectious to humans (Huang F F, et al. J Gen Virol, 85:1609-1618(2004)). The genomic RNA contains three ORFs (ORFs) encodingnonstructural proteins (ORF1), the viral capsid protein composed of 660amino acids (ORF2) and a small phosphorylated protein of unidentifiedfunction (ORF3) (Panda S K, Thakral D, Rehman S, Rev Med Virol,17:151-180 (2007); Tam A W, et al. Virology, 185:120-131 (1991)). Theviral capsid protein induces neutralizing antibodies by its immunization(Emerson S U, et al. J Gen Virol, 87:697-704 (2006); He S, et al., J GenVirol, 89:245-249 (2008); Meng J, et al., Virology, 288:203-211 (2001);Takahashi M, et al., Arch Virol, 153:657-666 (2008)) or during thecourse of infection (Schofield DJ et al., J Virol, 74:5548-5555 (2000);Schofield D J et al., Vaccine, 22:257-267 (2003)). A typical signalsequence at the N terminus and 3 potential N-glycosylation sites(Asn-X-Ser/Thr) are well-conserved in the capsid protein derived fromall mammalian genotypes (Graff J, et al., J Virol, 82:1185-1194 (2008);Zafrullah M et al., J Virol, 73:4074-4082 (1999)), but the glycosylationstatus of this protein is still controversial and the biologicalsignificance of the modification in the viral life cycle remainsunknown. Although propagation of HEV in the cell culture systemsreported in earlier studies was not efficient (Huang R, et al., ClinDiagn Lab Immunol, 6:729-733 (1999); Kazachkov Yu A, et al., Arch Virol,127:399-402 (1992); Meng J, Dubreuil P, Pillot J, J Clin Microbiol,35:1373-1377 (1997); Tam A W, et al., Virology, 238:94-102 (1997)),Tanaka et al. succeeded in the establishment of a persistent infectionsystem of HEV genotype 3 in human hepatoma (PLC/PRF/5) and humancarcinomic alveolar epithelial (A549) cell lines (Tanaka T et al., J GenVirol, 88:903-911 (2007)). However, sufficient amounts of viralparticles cannot be obtained for studies of the structure, life cycle,and pathogenesis of HEV.

Electron microscopy of human stool specimens showed that HEV is anonenveloped spherical particle with a diameter of approximately 320 Å(Bradley D, et al., J Gen Virol, 69:731-738 (1988)). As an alternativeto in vitro propagation of HEV, the baculovirus expression system opensthe prospect of studying HEV capsid assembly, since HEV-like particles(HEV-LP) with protruding spikes on the surface can be formed in insectcells infected with a recombinant baculovirus expressing the capsidprotein of a genotype 1 strain (Li T C, et al., J Virol, 71:7207-7213(1997); Li T C, et al., J Virol, 79:12999-13006 (2005); Xing L, et al.,Virology, 265:35-45 (1999)). Cryo-electron microscopic (cryoEM) analysishas revealed that HEV-LP is a T=1 icosahedral particle composed of 60copies of truncated products of ORF2 (Li T C, et al., J Virol,79:12999-13006 (2005); Xing L, et al., Virology, 265:35-45 (1999)). TheHEV-LP appeared to be empty due to a lack of significant densitycontaining RNA inside and was 270 Å in diameter (Li T C, et al., JVirol, 71:7207-7213 (1997); Li T C, et al., J Virol, 79:12999-13006(2005); Xing L, et al., Virology, 265:35-45 (1999)), which is smallerthan the diameter of the native virions. However, the HEV-LP retainedthe antigenicity and capsid formation of the native HEV particles.

The crystal structures of the recombinant or native T=3 viral particlesderived from structurally related mammalian and plant viruses, such asrecombinant Norwalk virus (rNV; PDB accession code 1IHM) (Prasad BV, etal., Science, 286:287-290 (1999)), San Miguel sea lion virus (SMSV; PDBaccession code 2GH8) (Chen Ret al., Proc Natl Acad Sci USA,103:8048-8053 (2006)), the members of the family Caliciviridae, andCarnation mottle virus (CARMV; PDB accession code 1OPO) (Morgunova E, etal., FEBS Lett, 338:267-271 (1994)), a member of the familyTombusviridae, have been determined at resolutions of 3.4 Å, 3.2 Å, and3.2 Å, respectively. In this study, to understand the structural basison HEV, we solved the crystal structure of HEV-LP derived from agenotype 3 strain at 3.5-Å resolution and found differences in thefolding of the capsid protein among these viruses. On the other hand, wefound several structural similarities of shell formation. In particular,it was revealed that aromatic amino acids (Tyr-288 in the case ofHEV-LP) at the 5-fold axis play a crucial role in the hydrophobicinteraction required for particle formation and are well conserved amongthese viruses. Furthermore, mutational analyses depicted the putativecellular receptor-binding regions and epitopes for neutralizing ofbinding (NOB) antibodies on the 3D structure of HEV-LP. The availabilityof the 3D structure of HEV-LP at high resolution will provide valuableinformation not only for analyses of the entry and assembly of HEV, butalso for the development of a vaccine for hepatitis E.

Results

Preparation of HEV-LP of a Genotype 3. Upon infection with a recombinantbaculovirus possessing a genome of the truncated capsid protein (aminoacids 112-608) from a genotype 3 strain under the control of polyhedrinpromoter, a large amount of HEV-LP was secreted into the culturesupernatant as described in the case of HEV-LP of genotype 1 strain (LiT C, et al., J Virol, 71:7207-7213 (1997); Li T C, et al., J Virol,79:12999-13006 (2005); Xing L, et al., Virology, 265:35-45 (1999)). Thepurified HEV-LP of genotype 3 was used for further structural andbiological analyses.

Overall Structure of HEV-LP. The crystal structure of HEV-LP derivedfrom the genotype 3 strain was determined at 3.5-Å resolution by themolecular replacement method by using a cryoEM map of HEV-LP derivedfrom the genotype 1 strain (Li T C, et al., J Virol, 79:12999-13006(2005); Xing L, et al., Virology, 265:35-45 (1999)) as an initialphasing model (FIG. 10A). As shown in the previous papers (Li T C, etal., J Virol, 79:12999-13006 (2005); Xing L, et al., Virology, 265:35-45(1999)), HEV-LP shows a T=1 icosahedral symmetry with an externaldiameter of 270 Å. This particle is composed of 60 subunits of thetruncated capsid proteins, forming the icosahedral 2-, 3-, and 5-foldaxes. It has 30 protrusions at the 2-fold axis of the surface with largedepressions at the 3- and 5-fold axes.

Structure of the HEV Capsid Protein. The truncated HEV capsid proteinhas 3 definite domains designated as S (shell), M (middle), and P(protruding) composed of the amino acid residues 129-319,320-455, and456-606, respectively (FIG. 10B). Because the N- and C-terminallytruncated capsid proteins were used for the characterization, thetypical signal sequence (amino acids 1-22) and following arginine-richdomain (amino acids 23-111) and the C-terminal domain removed bycleavage in insect cells (amino acids 609-660) were not determined inthis study. Additionally, the amino acid residues112-128,486-487,555-560, and 607-608 were disordered in this study. TheS domain, which forms an internal scaffold structure of the particle,folds into a classical anti-parallel jelly roll-like β-sandwichstructure with 8 β-strands (designated as B to I) and 4 short α-helicesthat are conserved among many viral capsids (FIG. 10B) (Prasad BV, etal., Science, 286:287-290 (1999); Chen R et al., Proc Natl Acad Sci USA,103:8048-8053 (2006); Morgunova E, et al., FEBS Lett, 338:267-271(1994); Hogle J M, Chow M, Filman D J, Science, 229:1358-1365 (1985);Tsao J, et al., Science, 251:1456-1464 (1991)). The M domain, which isone of the characteristic domains, has a twisted anti-parallel β-barrelstructure composed of 6 β-strands and 4 short β-helices. This domain istightly associated with the S domain and located on the surface aroundthe icosahedral 3-fold axis (FIGS. 10A and B). The M and P domains arelinked with a long proline-rich hinge (amino acids 445-467). Previousstudies on the structures of rNV (Prasad B V, et al., Science,286:287-290 (1999)) and SMSV (Chen Ret al., Proc Natl Acad Sci USA,103:8048-8053 (2006)) revealed that the P domains of the viruses arecomposed of 2 subdomains, P1 and P2, and the P2 subdomain is located asa large protrusion of the P1 subdomain (Fig. S1). In contrast, the Pdomain of HEV-LP is composed of a single individual domain forming atwisted anti-parallel β-sheets structure (FIG. 10B), demonstrating thatthe capsid protein of HEV-LP has a significantly different fold fromthose of caliciviruses, except for the S domain. Although we have noevidence of glycosylation of HEV-LP prepared in insect cells, the HEVcapsid protein has 3 potential N-glycosylation sites, Asn-137-Leu-Ser,Asn-310-Leu-Thr and Asn-562-Thr-Thr (Zafrullah M et al., J Virol,73:4074-4082 (1999)). In the dimer structure, the former 2 sites aremapped on the horizontal surface of the S domain, as shown in Fig. S2A.However, Asn-137 and Asn-310 are located in the interfaces of thepentamer and trimer structures, respectively (Fig. S2B and C),suggesting that, if it occurs at all, N-glycosylation in these sites mayinhibit assembly of HEV-LP. Indeed, Graff et al. (Graff J, et al., JVirol, 82:1185-1194 (2008)) reported that HEV carrying mutations inAsn-137 or Asn-310 to Glu lost infectivity to cells or rhesus macaquesdue to a defect in the virion assembly. On the other hand, Asn-562 ismapped in the central region in the top of the P dimer and exposed inthe surface of HEV-LP.

The Dimer Structure at the 2-Fold Axis. It is noteworthy that the HEV-LPdimer at the icosahedral 2-fold axis shows a crossing topology of the Pversus M and S domains, while that of the other viruses with protrusionsat the 2-fold axis, containing rNV, SMSV, and CARMV, exhibits a paralleltopology of each domain (FIG. 10C). The flexibility of the longproline-rich hinge region between the M and P domains allows this uniquetopology of HEV-LP. The P domain of HEV-LP interacts with not only the Pdomain but also the M domain of the counterpart to stabilize the dimerstructure. Despite these topological differences, the overall structureof the protrusion dimeric structure at the 2-fold axis is similar tothat of rNV and SMSV. The disordered residues 486-487 and 555-560 arelocated in the apical region of the protrusion, suggesting that thisregion is flexible to take advantage of the interaction with othermolecules.

Five-Fold Axis Packaging. The inter-molecule-interface of the capsidpentamer at the icosahedral 5-fold axis is composed of only S domains,and these interaction regions are narrower than those of the dimer andtrimer at the 2-fold and 3-fold axes, respectively (FIG. 11A),suggesting that the pentamer formation is a key step of HEV-LP assembly.There are 4 loops between the β-sheets in the S domain, designated asloops B-C (amino acids 139-152), D-E (amino acids 196-206), F-G (aminoacids 236-241), and H-I (amino acids 281-296), around the center of thepentamer structure. Among them, the loops B-C and F-G are not in closeproximity to the next subunits, suggesting they are not implicated inthe inter-molecular interaction. In contrast, loops D-E and H-I dointeract with the next subunits. In particular, the side chains ofAsn-200 and Tyr-288 in loops D-E and H-I, respectively, interact withthose of the next subunits, from which they are separated by a distanceof approximately 3.2 Å, filling in the central pore (FIG. 11A). Theseobservations led us to hypothesize that these amino acid residues areimportant for assembly and stability of the particles. To examine thishypothesis, we constructed 2 mutant capsid proteins in which Asn-200 wasreplaced with alanine (N200A) or Tyr-288 was replaced with alanine(Y288A), and the effect of these mutations on the particle formation wasdetermined by a density-fractionation assay (FIG. 11B). Comparativeamounts of the mutant proteins to the wild-type capsid were expressedand released into the supernatants of cells infected with therecombinant baculoviruses. N200A but not Y288A formed VLP as thewild-type, indicating that Tyr-288 plays a more crucial role in particleformation than Asn-200. The aromatic amino acids, Phe-118, Tyr-330, andPhe-145, are also found in the icosahedral 5-fold axis of rNV, SMSV, andCARMV, respectively (FIG. 11A). To examine the role of the aromatic sidechain in Tyr-288 in the particle formation, a series of mutants in whichTyr-288 was replaced with tryptophan, phenylalanine, leucine, asparaticacid, histidine, or arginine (Y288W, Y288F, Y288L, Y288D, Y288H, orY288R) were generated. All of them were expressed and released into theculture medium, as well as was the wild type. The mutants with aromaticamino acids, Y288W and Y288F, were able to form HEV-LP, whereas othermutants produced no or very few particles (FIG. 11B). These resultssuggest that the aromatic side chain of Tyr-288 plays a crucial role inthe HEV-LP formation by shutting off the central pore of the pentamer,and that the aromatic amino acids in the positions corresponding toTyr-288 of HEV are functionally conserved among the structurally relatedviruses.

Binding of HEV-LP to Cultured Cells. The early steps of HEV entry remainunclear because of the lack of a robust cell culture system for HEV,despite recent progress in the in vitro propagation of HEV in the celllines PLC/PRF/5 and A549 (Tanaka T et al., J Gen Virol, 88:903-911(2007)). HEV-LP was able to bind to several cell lines, includingPLC/PRF/5 and A549 cells, but not to mouse myeloma P3×63Ag8U.1 (P3U1)cells (Fig. S3), suggesting that a binding assay using HEV-LP is usefulto examine the first step of receptor-binding of HEV to the targetcells. Among the cell lines examined, the human hepatoma cell line Huh7,exhibited a greater ability to bind to HEV-LP than the cell linesPLC/PRF/5 and A549. Therefore, Huh7 cells were used for the followingbinding experiments of HEV-LP.

Three-Dimensional Mapping of Epitopes for NOB Antibodies. We examinedthe ability of the 10 newly produced anti-HEV-LP monoclonal antibodiesto inhibit the binding of HEV-LP to Huh7 cells (FIG. 12A). Two of themonoclonal antibodies, MAB1323 and MAB272, exhibited NOB of HEV-LP toHuh7 cells and recognized the P domain by immunoblotting using the GST(GST)-fused HEV capsid proteins. However, further truncation of theC-terminal 28 or N-terminal 24 amino acids from the GST-fused P domainabrogated the binding with the antibodies, indicating that it isdifficult to determine the epitopes of the antibodies in more detailusing a series of truncated mutants of the P domain. A competitiveenzyme-linked immunosorbent assay (ELISA) suggested thatMAB1323, MAB272,and MAB161, but not MAB358, which was used as a detector in the bindingassay, recognized the same or adjacent epitopes (Fig. S5). The P domainsof rNV and feline calicivirus were suggested to be involved in thebinding to the receptor molecules (Bhella D et al., J Virol,82:8051-8058 (2008); Bu W, et al. J Virol, 82:5340-5347 (2008); Choi J Met al., Proc Natl Acad Sci USA, 105:9175-9180 (2008)), and we thereforehypothesized that the P domain of HEV-LP might also be involved in thecell binding. To examine this possibility, we prepared 16 HEV-LP mutantsin which 1 or 2 amino acid residues at the surface of the P domain weresubstituted (FIG. 12B). The density fractionation assay indicated thatall of the mutant proteins formed HEV-LP in the manner of the wild-typecapsid protein. MAB358, which recognized an epitope on the M domain, wascapable of precipitating all of the mutants (FIG. 12C). MAB1323exhibited no interaction with mt3 and a weak precipitation of mt4 andmt12. Both MAB272 and MAB161 exhibited no or weak precipitation of mt5and mt15, whereas MAB272 but not MAB161 exhibited NOB of HEV-LP to Huh7cells (FIGS. 12A and C). The substituted amino acids of these mutantsare illustrated in the 3D structure of the capsid dimer (FIG. 13A), andthese results suggest that the NOB antibodies MAB1323 and MAB272recognize the peripheral region of the apical surface and the horizontalregion of the P domain above the M domain at the 3-fold axis,respectively.

Three-Dimensional Mapping of a Region Crucial for Binding to the TargetCells. To determine the region important for binding to the cellsurface, the mutant HEV-LPs substituted into the P domain were also usedin the assay of binding to Huh7 cells (FIG. 12D). The wild-type HEV-LPbound to Huh7 cells with a geographic mean fluorescence intensity (MFI)of 82.65. Among the mutants examined, mt4, mt11, mt12, and mt14exhibited significantly low MFI values of less than 20. Similar resultswere obtained using A549 cells. The amino acid residues required forcell binding were mapped in the central flexible region of the apicalsurface as shown in FIG. 13B. This region is partially overlapped withepitopes of MAB1323 (FIG. 13A) and other neutralizing antibodiesreported by Schofield et al. (Schofield D J et al., J Virol,74:5548-5555 (2000)) as shown in Fig. S7. These results suggested thatthe apical center region of the P domain is involved in the associationwith not-yet-identified cellular receptor(s).

Discussion

The expression of the truncated HEV capsid protein (amino acids 112-608)in insect cells resulted in assembly of HEV-LP, which retains anantigenicity similar to that of the native HEV particles (Li T C, etal., J Virol, 71:7207-7213 (1997); Li T C, et al., Virology, 349:222-229(2004)). This particle with a T=1 symmetry has a diameter of 270 Å,which is smaller than the 320-Å diameter of the native virion detectedin the fecal specimens of patients (Bradley D, et al., J Gen Virol,69:731-738 (1988)). It has been reported that the interior cavity ofHEV-LP is too small to accommodate a viral RNA of 7.8 kb in length (XingL, et al., Virology, 265:35-45 (1999)) and that the particles show noevidence of nucleotide contents (Li T C, et al., J Virol, 71:7207-7213(1997); Xing L, et al., Virology, 265:35-45 (1999)). Therefore, nativeHEV particles are suggested to be composed of a larger number and/or alarger size of capsid proteins than HEV-LP. In some cases of plantviruses with a T=3 symmetry, the capsid proteins assembled intoparticles with a T=1 symmetry by deletion of the N-terminal basic region(Hsu C, et al., Virology, 349:222-229 (2006); Kakani K et al., J Virol,82:1547-1557 (2008)) or amino acid substitutions either in theN-terminal region or in the linker domain between the N-terminal regionand S domain (Kakani K et al., J Virol, 82:1547-1557 (2008)), suggestingthat the N-terminal basic region plays an important role in switching ofthe transition from T=3 to T=1 symmetry. In addition, expression of theNV capsid protein in insect cells resulted in production of not only T=3large particles but also small particles thought to have the T=1symmetry (White L J, Hardy M E, Estes M K, J Virol, 71:8066-8072(1997)). Based on many similarities of the capsid structures and theirpackaging of structurally related viruses, the native HEV particles aresuggested to possess a T=3 surface lattice. The flexibility of theproline-rich hinge linking the M and P domains could allow the capsidprotein dimer to switch conformations between the A/B and C/C subunitsfound in T=3 viruses. Although structure of the native HEV may beslightly different from that of the HEV-LP, the data obtained in thisstudy by using HEV-LP should provide useful information to understandthe structure of viral particle, life cycle, and pathogenesis of HEV.The S domain shares the jellyroll fold with some other icosahedralviruses (Prasad B V, et al., Science, 286:287-290 (1999);Chen Ret al.,Proc Natl Acad Sci USA, 103:8048-8053 (2006); Morgunova E, et al., FEBSLett, 338:267-271 (1994); Hogle J M, Chow M, Filman D J, Science,229:1358-1365 (1985); Tsao J, et al., Science, 251:1456-1464 (1991)). Itwas found that the capsid proteins with substitutions of Tyr-288positioned at the center of the pentamer structure built in interSdomain-interaction failed to assemble into HEV-LP. Alignment analysis ofamino acid sequences using data available in GeneBank showed thatTyr-288 is completely conserved within 5 genotypes of HEV. Furthermore,residues corresponding to Tyr-288 of the HEV capsid protein are found inthe structures of rNV (Phe-118), SMSV (Tyr-330), and CARMV (Phe-145),although the positions of these aromatic residues are different. Tyr-288of HEV and Tyr-330 of SMSV located in the H-I loop and Phe-110 of rNV inthe D-E loop are exposed at the outside surface of the particles,whereas Phe-145 of CARMV located in the D-E loop is exposed at theinterior of the particle. These data suggest that the aromatic sidechains of these residues are involved in hydrophobic interactions withthose of the next subunits, assuring stable assembly of the particles.During entry into cells, rearrangement of the virion structure isrequired for release of the genome from the shell. However, the entryand uncoating mechanisms of HEV remain unknown. Because the center ofthe pentamer is the thinnest region of the particle and takes achannel-like structure (Xing L, et al., Virology, 265:35-45 (1999)),this region might also be important for uncoating and release of theviral RNA. It has been proposed that the 5-fold axis of poliovirus isinvolved in the genomic RNA translocation via conformational change ofthe virion initiated by binding to the receptor molecules (Belnap D M,et al. J Virol, 74:1342-1354 (2000); Bubeck D, Filman D J, Hogle J M,Nat Struct Mol Biol, 12:615-618 (2005)).

The first step in viral entry into a target cell is binding to thecellular receptors. The human hepatoma PLC/PRF/5 and lung epithelialA549 cell lines, which are highly susceptible to persistentHEV-infection (Tanaka T et al., J Gen Virol, 88:903-911 (2007)), arelikely to express functional HEV receptors on the cell surface. However,HEV-LP had reduced binding to these cells compared to the other celllines examined. Therefore, the human hepatoma cell line Huh7, which alsoexhibited a susceptibility to HEV infection (He S, et al., J Gen Virol,89:245-249 (2008); Graff J, et al., J Virol, 82:1185-1194 (2008)) andreadily bound to HEV-LP, was mainly used in this study. It has beenreported that the P domains of noroviruses and the feline caliciviruswere involved in the binding to the putative receptors, histo-bloodantigens (Bu W, et al. J Virol, 82:5340-5347 (2008); Choi J M et al.,Proc Natl Acad Sci USA, 105:9175-9180 (2008)) and the feline junctionaladhesion molecule (Bhella D et al., J Virol, 82:8051-8058 (2008)),respectively. The peptide of the HEV capsid protein (amino acids368-606), which consists of a part of the M and an entire P domain, wasshown to be capable of binding to several cell lines (He S, et al., JGen Virol, 89:245-249 (2008)), suggesting that the P domain of HEV isalso involved in the binding to the cell receptors. Indeed, themutational analyses in this study indicated that the central flexibleregion of the top of the P domain of HEV-LP plays a crucial role forbinding to Huh7 and A549 cells. This is consistent with a recent studyby Graff et al. in which an N562Q mutant of HEV lost infectivity toculture cells and rhesus macaques despite the production of viralparticles (Graff J, et al., J Virol, 82:1185-1194 (2008)).Interestingly, a possible N-glycosylation site, Asn-562-Thr-Thr, ismapped in this region. N-glycosylation is an unusual posttranslationalmodification for nonenveloped viruses, except for rotaviruses (JayaramH, Estes M K, Prasad B V, Virus Res, 101:67-81 (2004)). The mutantcapsid mt12, which has substitutions of Asn-562 and Thr-564 to alanine,exhibited the same migration as the wild-type protein in SDS/PAGE,suggesting that the HEV-LP produced in insect cells was not glycosylatedat Asn-562. Lack of N-glycosylation in the capsid protein has also beenreported in mammalian cells infected with HEV (Graff J, et al., J Virol,82:1185-1194 (2008)), whereas some portion of the capsid protein wasglycosylated and transported to the cell surface upon overexpression inmammalian cells (Zafrullah M et al., J Virol, 73:4074-4082 (1999)).N-glycosylation of the HEV capsid at Asn-562 may have a negative effecton the receptor-binding, whereas it may play a positive role in otherfunctions, including pathogenesis. The biological significance of theglycosylation of HEV capsid protein remains to be studied.

Although there is currently a lack of sensitive and reliable assays todetermine the neutralizing activity of anti-HEV antibodies, the assay ofNOB of HEV-LP binding to the target cells is thought to be a suitablealternative method. Measurement of the reactivity of a panel of mutantHEV-LPs revealed that the epitopes of MAB1323 and MAB272 antibodies aremapped in the peripheral region of the apical surface and the horizontalregion of the P domain dimer, respectively. These results furthersupport the notion that the P domain of HEV-LP is important for thebinding to cells. MAB1323 is suggested to directly inhibit theinteraction between HEV-LP and cellular receptors through binding to theapical surface, whereas MAB272 may have an allosteric effect, inducingconformational change of the P domain through binding to the horizontalregion. A number of monoclonal antibodies are capable of neutralizing invitro and in vivo infection of HEV (Emerson S U, et al. J Gen Virol,87:697-704 (2006); He S, et al., J Gen Virol, 89:245-249 (2008); Meng J,et al., Virology, 288:203-211 (2001); Takahashi M, et al., Arch Virol,153:657-666 (2008); Schofield D J et al., J Virol, 74:5548-5555 (2000);Schofield D J et al., Vaccine, 22:257-267 (2003)), and many of themrecognize conformational epitopes of the capsid protein, as seen in theMAB1323 and MAB272 antibodies prepared in this study. Monoclonalantibodies against linear epitopes located in amino acids 578-607 of agenotype 1 capsid protein (Schofield D J et al., J Virol, 74:5548-5555(2000)) were overlapped with a part of the putative receptor-bindingdomain and the epitope of MAB272, supporting the data of the presentstudy. On the other hand, monoclonal antibodies against the linearepitopes located in amino acids 423-438 and amino acids 423-443 in the Mdomain of a genotype 1 capsid protein neutralized binding of a peptidederived from the capsid protein to cells and HEV-infection (He S, etal., J Gen Virol, 89:245-249 (2008)), suggesting the importance of the Mdomain in the binding step.

In summary, we have determined the crystal structure of HEV-LP producedin insect cells and demonstrated its structural characteristics incomparison with the structurally related animal and plant viruses. Thisstudy will provide useful information for elucidation of the molecularmechanisms of HEV-life cycles and for the development of prophylacticand therapeutic measures for hepatitis E.

Materials and Methods

Expression, Purification, and Crystallization of HEV-LP. The recombinantbaculovirus encoding the ORF2 of the HEV genotype 3, 2712 strain wasexpressed in insect cells. HEV-LP was purified as described previously(Xing L, et al., Virology, 265:35-45 (1999)) and crystallized by thehanging-drop vapor-diffusion method. Details are reported in SIMaterials and Methods.

Data Collection and Phase Determination. x-ray diffraction data werecollected at 100 K on beamlines BL17A at the Photon Factory (KEK). Thestatistics of X-ray diffraction data collection are summarized inTable 1. The solved 3D structure of HEV-LP was submitted to the ProteinData Bank under the PDB accession code of 2ZTN. Details are reported inSI Materials and Methods.

TABLE 1 Data collection and processing statistics for HEV-LP Datacollection Space group P2₁2₁2₁ Cell dimensions a, b, c, Å 336.8, 349.4,359.5 X-ray wavelength, Å 1.0000 Resolution, Å   50-3.55 (3.68-3.55)R_(merge)* 0.131 (0.498) l/σl 9.8 (3.2) Completeness, % 99.9 (99.8)Redundancy 5.6 (5.2) Refinement Resolution range, Å 20-3.56 No.reflections 494, 466 R_(work)/R_(free) 30.5/30.9 No. atoms Protein 215,400 B factors Protein 94.9 rmsd Bond length, Å 0.009 Bond angle, ° 1.355Values in square brackets refer to the highest-resolution shell.*R_(merge)* = Σ_(hkl) Σ_(i)|l(hkl)_(i) − <l(hkl)>|lΣ_(hkl) l(hkl), wherel(hkl)_(i) is the ith measurement of the intensity of reflection hkl and<l(hkl)> is the mean intensity of reflection hkl.

Example 3 Structural Characterization of Predominant Antigenic Region onHEV Capsid Protrusion Domain

Hepatitis E virus (HEV) is a human pathogen that causes acute liverfailure. When expressed in insect cell with deletion of 52 amino acidfrom the C-terminus and 111 amino acids from the N-terminus, the capsidprotein can self-assemble into T=1 virus like particle (VLP) thatretains the antigenicity of native HEV virion. Here, using cryo-electronmicroscopy and image reconstruction, we determined that the anti-HEVmonoclonal antibodies bound to the protruding domain of the capsidprotein, with 60 copies of Fab fragment per VLP. The molecular dockingof HEV crystal structure revealed that the binding site of the antibodyMab224 covered three PORF2 surface loops at the rim of the surfaceplateau. This antibody binding site is separated from the potentiallocation of the inserted B-cell tag, an epitope of 11 amino acids fusedto the C-terminal end of the PORF2 protein, as it determined with thecryo-EM structure of chimeric HEV VLP. Therefore, the T=1 HEV VLP is arobust delivery candidate that induces effectively antibodies againstboth HEV and the foreign epitope.

Hepatitis E virus (HEV), a causative viral agent of acute liver failurein human, is primarily transmitted via fecal-and-oral route thusresistant to low pH and digestive enzymes associated with stomach anddigestive tracts. The infection of HEV can result in epidemic outbreaksin many tropic and subtropics countries. It is found that more than 50%of reported acute viral hepatitis cases are attributed to HEV in adultpopulation in India (Arankalle, V. A. et al., Proc Natl Acad Sci USA91:3428-32 (1994)) and 90% of jaundice hepatitis cases in the city ofKathmandu, Nepal in 1996 (Clayson, E. T. et al., Nepal. J Infect Dis176:763-6 (1997)). In India, 101 outbreaks were confirmed by serologicalanalysis in the state of Maharashtra during 2002-2007 (Deshmukh, T. M.et al., Vaccine 25:4350-4360 (2007)). The life risk of HEV infection inIndia is greater than 60% (Worm, H. C. Drugs 64:1517-1531 (2004)).Sporadic cases have also been reported between outbreaks in HEV-endemicregions as well as in non-endemic areas. Although some of these caseswere associated with travel to endemic regions, many cases involvedpatients without such a travel history. Accumulating evidence suggeststhat sporadic infection occurs through zoonotic routes, and hepatitis Ecases also prevalent in well-developed countries, including the UnitedStates (Purcell, R. H. et al., J Hepatol 48:494-503 (2008)). The overalldeath rates of HEV during outbreaks range from 1 to 15% in general andthe highest mortality occurs in pregnant women, with fatality rates ofup to 20% (Patra, S. et al., Ann Intern Med 147:28-33 (2007)). Moreover, HEV superinfection is documented to cause high rate of hepaticdecompensation (Kumar, A. S. et al., J Hepatol 46:387-394 (2007)) andprogression of acute hepatitis E to chronical liver disease is alsoreported in the organ-transplant recipients (Kamar, N. et al., N Engl JMed 358:811-817 (2008)).

The HEV virion is composed of a single-stranded RNA molecule of 7.2 kBin size and an icosahedral capsid reported to be 32-34 nm. The HEVcapsid protein of 660 amino acids (ORF2 protein) is encoded by thesecond open reading frame and is responsible for most capsid-relatedfunctions, such as assembly, host interaction, and immunogenicity.Recombinant ORF2 protein can induce antibodies to prevent HEV infectionin non-human primates (Li, T.-C. et al., Vaccine 22:370-377 (2004);Purcell, R. H. et al., Vaccine 21:2607-15 (2003); Tsarev, S. A. et al.,Vaccine 15:1834-1838 (1997)). Four major antigenic domains werepredicted to be located in the C-terminal 268 amino acids of ORF2proteins (Meng, J. et al., Virology 288:203-211 (2001)), one of whichwas experimentally identified as the neutralization epitope on theSar-55 ORF2 capsid protein (Schofield, D. J. et al., J Virol74:5548-5555 (2000)). A truncated ORF2 peptide containing residues459-607 of ORF2 protein presents the minimal peptide needed to induceanti-HEV neutralizing antibody (Zhou, Y. H. et al., Vaccine 23:3157-3165(2005)), suggesting that the HEV neutralizing epitope isconformation-dependent. Currently, there are 1,600 genomic sequences ofHEV available at the International Nucleotide Sequence DatabaseCollaboration, which are grouped into four genotypes. Notably, only asingle known serotype is recognized and the antibodies from any one ofthe four genotypes broadly cross-reactive with genotype-1 HEV virus,suggesting that the immuno-dominant domain of HEV is highly conserved.(Emerson, S. et al., J Gen Virol 87:697-704 (2006)).

Like other hepatitis viruses, HEV is unable to propagate in largequantities in current cell culture systems. Development on HEVpreventive strategies rely on recombinant protein derived from the HEVcapsid protein. When expressed in insect cell, the recombinant ORF2protein self-assembles into virus-like particles (VLPs) after deletionof 52 residues from the C-terminus and 111 residues from the N-terminus(PORF2) (Li, T. C. et al., J Virol 71:7207-7213 (1997)). Our previousstructural analysis of recombinant HEV-VLP by cryo-electron microscopy(cryo-EM) provided a basic understanding of the quaternary arrangementof PORF2, where the reconstructed VLP displayed a T=1 icosahedralparticle composed of 60 copies of truncated PORF2 (Xing, L. et al.,Virology 265:35-45 (1999)) and the essential assembly element of PORF2protein contained amino acids 125-600 (Li, T.-C. et al., J. Virol.79:12999-13006 (2005)). Recently, crystal structures were reported forgenotype-3 T=1 VLP (Yamashita, T. et al., Proc Natl Acad Sci USA106:12986-12991 (2009)) and genotype-4 T=1 VLP (Guu, T. et al., ProcNatl Acad Sci USA 106:12992-12997 (2009)), which revealed that PORF2 iscomposed of three domains. Although these VLPs (270 Å in diameter) issmaller than the native HEV virion (320-340 Å), this HEV-VLP can induceanti-HEV antibody when orally administered to experimental animals (Li,T. et al., Vaccine 19:3476-3484 (2001)).

There is a need of structural information on HEV antigenic epitopes forthe development of preventive strategies. Here, we present a series ofexperiments using cryo-EM and three-dimensional reconstruction toidentify the antigenic structure. Our results indicate that theantibody-binding footprint covered the lateral side of the P domain andseparated from the location of the inserting B-cell tag.

Materials and Methods

Production and Purification of Anti-HEV Monoclonal Antibodies

Eight-week-old female BALB/c mice were immunized at 0 and 4 weeks byintraperitoneal inoculation with HEV VLPs (100 ug/ml). Four weeks later,a final boost of equal volume of antigen was administered. Three daysafter the final boost, mouse spleen cells were fused with P3U1 mousemyeloma cells using polyethylene glycol 1500 (50% [w/v]) (BoehringerMannheim, Germany) as essentially described previously (Adler, B. etal., Pathology 15:247-250 (1983)). Supernatant from microplate wellspositive for hybridoma growth was screened by ELISA using therecombinant HEV VLPs as antigen. Hybridomas secreting specificantibodies to HEV were subcloned three times by limiting dilution, afterwhich they were considered to be monoclonal. Antibodies in thesupernatants were isotyped using the Mouse Monoclonal Antibody Isotypingkit (Amersham, Little Chalfont, Buckinghamshire, U.K.) in accordancewith the manufacturer's protocol. Hybridomas were grown in bulk instationary flasks (Nunc, Roskilde, Denmark) using PRMI-1640 with 15%FCS. Supernatant was harvested and antibodies were purified using HiTrapprotein G affinity columns (Pharmacia Biotech AB, Uppsala, Sweden) andstored at −80° C. Among all the antibodies, Mab224 used in our analysisis immunoglobulin G1 (IgG1) isotype.

Preparation of Fab Fragments

Papain cleavage was used to prepare isolated Fab fragments. A reducingL-cystein buffer was used to activate papain, and the MAb224 was mixedwith papain at a molar ration of 100:1. The mixture was incubatedovernight at 30° C. The reaction was quenched by adding iodacetamide andproduct was analyzed on SDS-PAGE. The Fab fragments were purified usinga Protein-A column according to the manufacturer's instruction. The Fcfragments and uncleaved Mab224 were trapped in the column due to theaffinity to protein-A while the Fab fragments were collected in theflow-through fractions.

Preparation and Purification of the HEV VLPs

The production and purification of HEV VLPs were performed according tothe protocol described earlier (Li, T. C. et al., J Virol 71:7207-7213(1997); Niikura, M. et al., Virology 293:273-280 (2002)). Briefly, theDNA fragments containing the N-truncated ORF2 protein (for wild VLP),and chimeric ORF2 protein (for VLP/C-tag) were cloned with baculovirustransfer vector pVL1393 to yield pVLORF2. The recombinant baculoviruseswere produced from Sf9 insect cells (Riken Cell Bank, Tsukuba, Japan)and then infected Tn5 insect cell at a M.O.I.>5. The infected insectcells were incubated in EX-CELL™ 405 medium (JRH Biosciences, Lenexa,Kans.) for 6 days at 26.5° C. The culture medium was collected afterremoval of cell debris by centrifugation at 10,000 g for 90 min. Thesupernatant was spun down at 30,000 rpm for 2 h in a Beckman SW32 Tirotor and pellet was resuspended in 4.5 ml EX-CELL™ 405. The pelletcontained HEV VLP and was further purified within CsCl density gradient.The white virus-band was collected and diluted 4 times with EX-CELL™ 405to remove CsCl by centrifugation (2 h in a Beckman TLA 55 rotor at45,000 rpm). The VLPs were re-suspended in 100-500 μl of 10 mMpotassium-MES buffer and stored at 4° C. To construct chimericVLP/C-tag, the recombinant baculoviruses was prepared by insertingB-cell tag epitope from glycoprotein D of herpes simplex virus“QPELAPEDPED” (SEQ ID NO:7) after residue 608 (Schofield, D. J. et al.,Vaccine 22:257-267 (2003)). The VLP/C-tag is prepared and purified withthe protocol described above.

Preparation of VLP-Fab Complexes for Cryo-Electron Microscopy

The VLP/Fab complexes were prepared by incubating Fab fragments with VLPat a molar ratio exceeding 1:300 at 4° C. over night. Unbound Fabfragments were removed by running the sample through a shortgel-fitration column containing Sephacryl-300. The fractions containingVLP/Fab complexes were identified based on reading of OD280 nm. The Fabbinding occupancy was roughly estimated by SDS-PAGE, in which thepurified VLP/Fab complexes were loaded on an acrylamide gel (gradient8-25%) and electrophoresis was run on a Phast™ system (Pharmacia) underconstant-voltage condition. The integrity of particles was checked byelectron microscopy (EM) after negatively stained with 2% uranyl acetate(UA).

Cryo-electron Microscopy

The sample preparation and cryo-EM data collection were followed awell-established procedures described previously (Xing, L. et al.,Virology 265:35-45 (1999)). Briefly, a drop containing 3.5 μl of samplewas applied on a glow-discharged homemade holey-carbon grid, blottedwith a piece of filter paper to remove the extra liquid and quicklyplunged into liquid ethane cooled by liquid nitrogen. Thefrozen-hydrated specimen grid was then transferred into a FEI CM-120electron microscope with a Gatan 626DH cryo-holder which kept specimenat the low temperature environment (−178° C.) for the subsequent datacollection. Micrographs were recorded under low-dose condition (<10e-/Å²) on Kodak SO163 films at a magnification of 45,000× with thedefocus range from 1000 to 3000 nm. Micrographs were visually selectedbased on the criteria of suitable particle concentration, optimal icethickness and minimal specimen drift. Only those micrographs fulfillingthese criteria were analyzed.

Image Processing

Selected micrographs were digitized using a Heidelberg PrimescannerD8200 (Heidelberg, Germany) at a 14 μl scanning step size, correspondingto 3.11 Å per pixel at specimen space. Particles were manually pickedusing Robem (v2.14), and centered by cross-correlating each of themagainst the circular average image. The astigmatism and the defocusvalue of each microsgraph were evaluated by the superimposed the powerspectra from all picked particles within the micrograph. The images usedfor the structural determination have their first zero approximatelylocated within the range of 17-20 Å⁻¹. The self-common lines algorithmwas used to yield the initial models for VLP/C-tag, VLP/Mab224 andVLP/Mab4, respectively (Crowther, R. A. Philos Trans R Soc Lond B BiolSci 261:221-230 (1971)). The refinement on particle origin andorientation was carried out iteratively using polar Fouriertransformation algorithm (PFT) (Baker, T. et al., J. Struct. Biol.116:120-130 (1996)). Three-dimensional reconstructions were computed bycombining a set of particles with the orientations evenly distributed inan icosahedral asymmetric unit, with the Fourier-Bessel algorithm whilesuperimposing of 5-3-2 icosahedral symmetry. To examine the reliabilityof the three-dimensional reconstruction, the dataset was evenly dividedinto two parts and computed two three-dimensional reconstructions. Theresolution was estimated using Fourier shell correlation (FSC) byassessing the agreement between these two reconstructions in the Fourierspace; a coefficient value of 0.5 was used as the criteria to estimatethe effective resolutions of VLP/C-tag, VLP/Mab224, and VLP/Mab4 at 17.5Å, 18.5 Å, and 24 Å respectively.

The electron density map was displayed in the isosurface mode, whichbuilds a surface barrier to contour the density about a certainthreshold to provide the concise surface details on the density map. Thecontour level was chosen at the value corresponding to the 100% mass ofthe particle volume. The surface presentations of the cryo-EM densitymaps was prepared with the program Chimera (Pettersen, E. F. et al., JComput Chem. 25:1605-1612 (2004)).

Fitting the Crystal Structure into Cryo-EM Density Maps

Manually fitting was carried out by translational and rotationalmovement of the PORF2 crystal structure into the cryo-EM density mapsusing program 0 (Jones, T. A. et al., Acta crystallogr. Sect. A47:110-119 (1991)) To get the best fit, the coordinates of PORF2 subunitwas treated as a rigid body. To optimize the fitting results,symmetry-related molecules were generated and judged by the crashesbetween subunits. The fitting was evaluated based on the crosscorrelation coefficient (cc value) between the cryo-EM density and thedensity computed from the fitted PORF2 coordinates. The fitting wasconsidered as stable when the cc value reaching 80%. The figures onfitting were prepared using the program PyMOL (DeLano, W. L. DeLanoScientific, Palo Alto, Calif., USA (2002)) and the surface stereographicprojection of the HEV VLP was prepared with the program RIVEM (Xiao, C.et al., J Struct. Biol. 158:181-186 (2007)).

Results

Binding of Antibody Mab224 Relies on the Presence of Residues 601-608Protein

The binding of monoclonal antibody Mab224 to PORF2 was characterizedwith a Western Blot assay because no cell culture system is currentlyavailable to test the ORF2 escape mutants. A series of C-terminallytruncated PORF2 proteins were separated using 10% SDS-PAGE underreducing condition, and blotted with Mab224 (FIG. 1A). Mab224 recognizedPORF2-derived peptides containing regions of 112-660, 112-608, 112-602and 112-601. In contrast, the peptides comprising residues 112-600,112-596, and 112-589 were non-reactive with Mab224. These data suggestthat residue Leu601 is critical for Mab224 binding with the PORF2protein. The lower molecular weight bands detected by the Mab224 aredegraded ORF2 proteins that contain the binding sequence of the Mab224.

Two-dimensional Electron Cryo-micrographs

On cryo-electron micrographs, both chimeric HEV particles andFab-conjugated VLP complex had circular profiles with spiky densitiesextending from the surface (FIG. 1C). They appeared as empty particleand similar to the morphology we observed previously with the HEV nativeVLPs (Li, T.-C. et al., J. Virol. 79:12999-13006 (2005); Xing, L. etal., Virology 265:35-45 (1999)), indicating that RNA moiety is absencefrom these VLPs. The sizes of both VLPs were approximately 27 nm withouttaking into account the extra densities extending further from thesurface of VLP/Mab224 (FIG. 1C, right).

Binding Footprints of the Antibodies

The cryo-EM structure of VLP/Mab224 was reconstructed from 615 imagesand displayed T=1 icosahedral symmetry with 60-subunits per particle.There are 30 dimeric protruding spikes located at each icosahedraltwo-fold axis (FIG. 14A). Sixty Fab fragments were observed around eachVLP particle and jutted from the lateral sides of the P domain. The Fabdensity was measured as ˜57 Å high from the surface of the spike. Thedensity corresponding to Fab fragment was approximately equal inmagnitude to that of the HEV VLP, indicating that most or all of the 60binding sites had been occupied by a Fab molecule. Using the density mapof HEV VLP (Xing, L. et al., Virology 265:35-45 (1999)), the densitycorresponding to VLP capsid was removed from the cryo-EM map, producinga Fab difference density map that was used to pinpoint the binding siteof Mab224 antibody (footprint).

Meanwhile, we determined the structure of HEV VLP in complexed with aneutralizing antibody, Mab4 by combining 234 individual images. By invitro immunoprecipitation, Mab4 is found to precipitate native HEVvirion and the recombinant PORF2 peptide, but does not react with thepeptides missing amino acids 597-607 (Schofield, D. J. et al., Vaccine22:257-267 (2003)). The three-dimensional reconstruction of VLP/Mab4complex showed sixty copies of Fab fragment per HEV VLP, indicating oneFab fragment per PORF2 subunit. Unlike the complex of Mab224, thedensity corresponding to Mab4 was about one third in magnitude to thatof the capsid (FIG. 14A), suggesting that only 30-40% of the bindingsites were occupied by the Fab fragments. More over, the Fab differencemap indiates that the Fab fragment of Mab4 appeared to contact withthose residues at the spike stem region, as a result, the density abovethe spike surface represented only the Fc domain (FIG. 14A). Both Mab224Fab fragment and Mab4 Fab fragment extends along the long axis ofprotruding P domain and appeared no steric hindrance with theneighboring Fab molecules at the regions around five-fold axis and thataround three-fold axes. The density profile of bound Fab fragmentsappeared 90° different at radius corresponding to the spike surface (135Å). The long axis of Mab224 extends toward neighboring spike, while thelong axis of Mab4 pointed to the fivefold axis (FIG. 14B).

To further analysis the binding interface of Fab and HEV VLP, we dockedthe crystal structure of genotype-1 PORF2 into the cryo-EM density mapof VLP/Mab224. The crystal structure of genotype-1 PORF2 was composed ofthree domains and in good agreement with those of genotype-3 andgenotype-4 PORF2 (PDB accession number 2ZTN and 3HAG, respectively)(Xing, manuscript in preparation). The coordinates fitted very well withthe cryo-EM density map without any adjustment. The Fab fragment ofMab224 interacted with the residues at side of the ORF2 spike ratherthan those residues on the plateau surface of the spike (FIG. 15A). Thecontact footprint does not overlap with the dimeric interface of thePORF2 spike. As we expected, Mab224 is a conformational antibody, whosebinding site covers three surface loops: the AB loop (470-493), the CDloop (539-569) and the EF loop (581-595) (FIG. 15B). The residues E479,D481, T484, Y485, S487 from the AB loop and residues Y532, S533, andK534 from the CD loop were in close contact with the Fab molecule.

Structure of HEV Chimeric VLP

The PORF2 fusion protein was constructed to incorporate a B-cell tag of11 amino acids to the C-terminus of PORF2 (FIG. 1 c), from which thechimeric HEV VLP (VLP/C-tag) was assembled. A total of 782 images ofindividual particles were used to reconstruct the finalthree-dimensional model of VLP/C-tag. In agreement with the previouslypublished cryo-EM structure of VLP, the surfaces of VLP/C-tag can alsobe divided into two distinct layers, an icosahedral shell and aprotruding spike (FIG. 16A). The spike projects outward from theicosahedral shell and composed of a PORF2 dimer. The distance betweeneach two-fold axis was ˜76 Å, measured between the top surfaces of twoadjacent P domains, which is consistent with the results measured in theVLP obtained from Tn5 insect cells and that from Sf9 insect cells. Nosignificant RNA density was found within the chimeric VLP/C-tag.

The crystal structure fit very well with the density map of VLP/C-tag(FIG. 16B), indicating that the C-terminal insertion of 11 amino acidsinhibits neither the dimer-dimer interactions nor the formation of T=1VLP. When superimposing the density maps at the contour covering 100%protein mass, we found that the radii of the S domain were roughly thesame in both VLP/C-tag and VLP/Mab224 maps and the height of protrudingspike appeared similar. No significant density difference was observedon the surface of spike plateau from the docking (FIG. 17), suggestingthat the inserted B-cell tag is flexible and less ordered. However,there appeared extra density at lateral side of the spike and underneaththe Mab224 binding site (FIG. 17A), which may corresponding to theinserted peptides.

Discussion

The HEV T=1 VLP is a recombinant virus-like particle that inducesanti-HEV antibody in non-human primates (Li, T.-C. et al., Vaccine22:370-377 (2004)). It can also be used as antigen carrier to deliverforeign epitopes through oral administration (Niikura, M. et al.,Virology 293:273-280 (2002)) and DNA vaccines (Takamura, S. et al., GeneTher 11:628-635 (2004)). Therefore, the structural analysis on antibodyrecognition sites is essential for developing VLP based anti-viralstrategies. For this purpose, we determined the structure of HEV VLP incomplexed with antibody Mab224 (VLP/Mab224) and antibody Mab4 (VLP/Mab4)as well as the structure of chimeric HEV VLP carrying a B-cell tag atPORF2 C-terminus (VLP/C-tag). The docking of PORF2 crystal structureprovides us the spatial information on HEV antigenic domain andstructural guidance to better design the insertion of foreign epitopes.

Structure of the Neutralization Epitopes

Antigenic properties and neutralization mechanism in HEV are difficultto be characterized due to the fact of lacking adequate replicationsystems in cell cultures. Therefore, our understanding of HEV immunologyare mainly based on the studies using recombinant proteins expressed inEscherichia coli (E. coli) and recombinant proteins or HEV-VLPs ininsect cells. All the evidences indicated that the C-terminal region ofPORF2 participates in the immune response of HEV and suggested that theneutralization epitope of HEV is conformational. Later, the minimumpeptide required to induce HEV neutralizing antibody was identified to148 residues of PORF2, from amino acids 459-607 (Zhou, Y. H. et al.,Vaccine 23:3157-3165 (2005)). This peptide region coincides with theP-domain revealed in the crystal structures of PORF2. Our cryo-EMstructures revealed that the Fab fragment attached entirely with thespikes, thus experimentally demonstrated that the P domain carriesprimarily the HEV antigenicity. Mab4 is a chimpanzee antibodies againstthe ORF2 protein isolated against with phage display from a coda libraryof chimpanzee (Schofield, D. J. et al., J Virol 74:5548-5555 (2000)). Itbinds to native HEV virion and the recombinant PORF2 peptides containingamino acids 597-607 (Schofield, D. J. et al., Vaccine 22:257-267(2003)). We did fitting with the structure of HEV/Mab4, however, thedensity of Mab4 is too weak to provide conclusive binding orientation ofthe Mab4, although the density corresponding to the Fab fragment didcover the location of amino acid 606 (data not shown). It is not clearwhy Mab224 appeared non-reactive with the peptides lacking amino acids601-608 on Western Blot, however, the binding site of Mab224 isconsistent with the critical antigenic residues determined earlier withmutagenesis. It is found that the point mutations of charged residuesE479, K534 to alanine as well as the hydrophobic amino acids Y485 andI529 to alanine would selectively abrogate the reactivity with theneutralizing antibodies (Li, S. et al., PLos Pathog. 5:e1000537 (2009)).Another set of mutants suggested the same region as antibody recognitionresidues spreading over the loops of AB, CD and EF (Yamashita, T. etal., Proc Natl Acad Sci USA 106:12986-12991 (2009)). This neutralizationsite is partially overlapped with the receptor binding site, where thebound antibody create spatial hindrance to prevent HEV VLP fromattachment to the cell surface (Yamashita, T. et al., Proc Natl Acad SciUSA 106:12986-12991 (2009)). The antibodies used in both experiments arewith neutralizing activity; therefore, the monoclonal antibody Mab224 ismost likely a neutralizing antibody because its binding site is part ofHEV dominant neutralization surface.

Insertion Sites for Foreign Epitopes

Virus-like particles (VLPs) are one of the robust cargoes to carrysimultaneously small molecules, peptide antigenic epitopes, as well asDNA vaccines of heterogonous sources in targeting other diseases becausethey are highly organized capsules that mimic the overall structure ofvirus particles. This approach relies on excellent structuralinformation of VLPs that allows rational design on where the foreignepitopes are conjugated. Six insertion sites were previously selected onPORF2 without knowing the crystal structure. They are the N- and theC-termini and four internal sites. The internal sites are after residuesA179, R366, A507, and R542. The fusion proteins carrying insertion atsites A179 and R336 failed completely in VLPs production and thosecarrying insertion at A507 and R542 greatly reduced VLP productionwithout affecting peptide expression (Niikura, M. et al., Virology293:273-280 (2002)). The crystal structure revealed that these internalinsertions are at the wrong spatial positions. The residue A179 islocated in the S1 domain in the middle of α-helix while the completenessof the α1 helix is necessary for the integrity of S1 domain thus theinteraction with its twofold related neighboring subunit (FIG. 16C).R366 is located in the S2 domain and favors electrostatic interactionwith the residue E386 from its threefold related neighboring subunit(FIG. 16D). Although located at P domains, the side chain of R542 iswithin the dimeric interface and guides the hydrophobic interaction oftwo monomers (FIG. 16D). Insertion after R542 may misalign theorientation two P domains that weaken the dimeric interaction of PORF2protein. The role of residue A507 in the P-domain is to maintain the Pdomain orientation by fixing the angle to the long proline-rich hinge(FIG. 16C). None of the four residues are exposed on the surface of VLP,although some of them are at the surface of individual PORF2 subunits(FIG. 16). Therefore, insertion of a foreign sequence at these sitesinduces no interference to the expression of individual protein, butdoes bring in hindrance to the assembly of HEV VLPs. The crystalstructure revealed that the C-terminus is exposed on the surface of VLPand N-terminal is pointing toward the VLP center. Therefore, insertionat these two sites does not inhibit the assembly of VLP; however, theC-terminus is more suitable to tether bulky foreign antigenic sequences,as it exposed on the VLP outer surface (FIG. 17).

The cryo-EM structure of the chimeric HEV VLP/C-tag suggested thelocation of the B-cell tag at the lateral site of the spike, not farfrom the residue A606 (C-terminal end) (FIG. 17A). This densitysaturated beneath the binding site of Mab224, but overlapped with thepotential binding site of Mab4. As a result, the insertion of B-cell 11amino acids may open partially the HEV antigenic site to host immunesystem. This explains why the infected mouse can develop antibodiesagainst both HEV and the foreign epitope after oral administrated withthe chimeric VLP/C-tag.

In conclusion, insertion of foreign epitope on the C-terminus of PORF2does not intervene with the accessibility of HEV antigenic domain.Therefore, the chimeric HEV VLP is able to induce antibodies againstboth HEV and the target disease. As recombinant ORF2 VLP is currentlyunder phase II clinic vaccine trial (26), the T=1 HEV VLP will play animportant role in oral delivery.

Example 4 Structural Basis for the RNA-Dependent Assambly Pathway ofHepatitis E Virion-Sized Particles

Hepatitis E virus (HEV) induces acute liver failure in human with highfatality rate in pregnant women. There is a need for anti-HEV researchto understand the assembly process of HEV native capsid. Here, weproduced a large virion-sized and a small T=1 capsid by expressing theHEV capsid protein in insect cells with and without the N-terminal 111residues, respectively, for comparative structural analysis. Thevirion-sized capsid demonstrates a T=3 icosahedral lattice and containsRNA fragment in contrast to the RNA-free T=1 capsid. However, bothcapsids shared common decameric organization. The in vitro assemblyfurther demonstrated that HEV capsid protein had intrinsic ability toform decameric intermediate. Our data suggest that RNA-binding is theextrinsic factor essential for the assembly of HEV native capsids.

Hepatitis E virus (HEV), the causative agent of acute hepatitis inhuman, is primarily transmitted through contaminated water and generallyresults in epidemic outbreaks in many developing countries. Sporadiccases have also been reported between outbreaks in HEV-endemic regionsas well as in non-endemic areas and these cases are transmitted throughzoonotic route. The overall mortality rates of HEV during outbreaksrange from 1 to 15% in general and the highest mortality occurs inpregnant women, with fatality rates of up to 30% (Naik, S. R. et al.,Bull World Health Organ 70, 597-604 (1992)).

HEV consists of a non-enveloped icosahedral capsid and asingle-stranded, positive-strand RNA genome of ˜7.2 kb that encodesthree open reading frames (ORFs) (Tam, A. W. et al., Virology 185,120-131 (1991)). The capsid protein, encoded by the ORF2, is composed of660 amino acids and responsible for most capsid-related functions, suchas virion assembly, host interaction, and immunogenicity. Like otherhepatitis viruses, HEV is unable to propagate in currently availablecell culture system and the research of HEV relies largely on therecombinant HEV capsid proteins (Schofield, D. J. et al., Vaccine 22,257-267 (2003); Li, T.-C. et al., Vaccine 22, 370-377 (2004); Purdy, M.A. et al., J Med Virol 41, 90-94 (1993); Riddell, M. A. et al., J Virol74, 8011-8017 (2000)). Virus-like particle (VLP) was obtained when thetruncated HEV capsid protein was expressed in insect Tn5 cells withdeletion of 52 residues from the C-terminus and 111 residues from theN-terminus (PORF2) (Li, T. C. et al., J Virol 71, 7207-7213 (1997)). Ourprevious structural analysis of this HEV-VLP by cryo-electron microscopy(cryo-EM) provided a basic understanding of the quaternary arrangementof PORF2, where the reconstructed VLP displayed a T=1 icosahedralparticle composed of 60 copies of PORF2 (Xing, L. et al., Virology 265,35-45 (1999)). The essential element of PORF2 protein for T=1 VLPassembly includes amino acids 125-600 (Li, T.-C. et al., J. Virol. 79,12999-13006 (2005)). Recently, the structural information was furtherrefined by the crystal structures of genotype-3 T=1 VLP (Yamashita, T.et al., Proc Natl Acad Sci USA 106, 12986-12991 (2009)) and genotype-4T=1 VLP (Guu, T. et al., Proc Natl Acad Sci USA 106, 12992-12997(2009)), which revealed the tertiary structure of PORF2 to the level ofamino acids. However, the T=1 VLPs used in these experiments were muchsmaller than that of the native virion, which has a diameter of 320-340Å, as determined by immuno-EM (Balayan, M. et al., Intervirology 20,23-31 (1983)). There is still a need to investigate the assembly pathwayof HEV capsid.

We previously hypothesized that HEV virion could be made of 180 copiesof the capsid protein (Xing, L. et al., Virology 265, 35-45 (1999)). Totest this hypothesis, we screened for HEV genotype expression andsuccessfully produced a virion-sized VLP from the HEV genotype-3 ORF2protein after deleting 52 residues from C-terminus. This VLP allowed usto investigate the molecular interactions that govern HEV virionassembly.

Experimental Procedures

Production of HEV-VLPs and in Vitro Disassembly and Reassembly

HEV-VLPs were produced and purified according to the protocol describedpreviously (Li, T. C. et al., J Virol 71, 7207-7213 (1997)). Briefly,the recombinant baculovirus is constructed to encode genotype-3 HEV-ORF2protein from residue 14 to 660 (Li et al., manuscript in preparation).Tn5 cells were infected with recombinant baculovirus at an M.O.I of fiveand cultured for 6 days. The supernatant was collected and the VLP waspurified by multiple ultracentrifugations, followed by separation on aCsCl density gradient. The final pellet was resuspended in 10 mMpotassium-[2-(N-morpholino)ethanesulfonic acid] (MES) buffer, pH 6.2. Ahomemade dialysis device was used in the disassembly and reassemblyexperiments, because it allowed dialysis with a small amount of sample(20-40 μl). Purified VLP was disrupted by dialysis against buffercontaining EDTA (10 mM) and DTT (20 mM) at different pHs. After VLPdissociation, 150 mM NaCl in Tris-HCl buffer (pH 7.5) was added, andsample was examined under the electron microscope after one-hourincubation in the presence of the divalent ion Ca²⁻ (20 mM).

Scanning Transmission Electron Microscopy Analysis of HEV-VLPs

Scanning TEM was performed at the Brookhaven National Laboratory STEMfacility, with TMV as an internal control. The mixture of VLP and TMVwas quickly frozen in liquid nitrogen, and then maintained at −150° C.during data collection to eliminate contamination and reduce mass loss.The specimen was scanned by a 40 keg electron beam of 0.25 nm in size,and images were collected with a preamp gain of 10 for both large andsmall angle detectors (Wall, J. S. et al., Methods Cell Biol. 53,139-164 (1998)). The image was recorded with a pixel size of 10 Å andwas analyzed with the PCMass29 program (Brookhaven National Labhttp://www.biology.bnl.gov/stem). After normalizing the background, themass of the VLPs was selected with the MSV shell model provided by theprogram. Mass measurements for TMV and HEV-VLPs were always performedfrom the same image. The HEV-VLP mass was measured in MDa (mass perparticle) and the TMV mass was measured in KDa/Å (mass per unit length)(Wall, J. S. and Simon, M. N., Methods Mol Biol. 148, 589-601 (2001)).

Cryo-electron Microscopic Structure Determination of HEV T=3 VLP

The collection of cryo-EM data for image reconstruction was performed ona JEOL JEM-2100F TEM operating at 200 kV according to the proceduredescribed in detail previously (Xing, L. et al., Virology 265, 35-45(1999)). Briefly, a 3 μl solution containing E330K capsid or reassembledORF2 complex was placed on holey carbon film-coated copper grids, andthen quickly plunged into liquid ethane after the removal of excesssolution. The VLPs were embedded into a thin layer of vitrified ice andtransferred into the EM using a Gatan 636 cryo-transferring system. Thespecimen was observed under 50,000× magnification and the area ofinterest was recorded on a TVIPS CCD camera (TemCam-F415). Themicrographs were recorded with a pixel size of 2.0 Å at a specimen spaceand defocus level of 0.7-3.5 Å (FIG. 26 a). Digital images with nostigmatism or drift were selected for later image processing. Images ofindividual HEV T=3 VLP were then boxed out and processed with anestablished software package for icosahedral particles (Baker, T. S.,and Cheng, R. H. (1996) J Struct Riot 116, 120-130; Ji, Y. et al., JStruct Biol 154, 1-19 (2006)). In total, 7720 individual images wereincluded in the process, and their defocus levels were distributedmainly within 1.0-2.5 Å.

To correct CTF (contrast transfer function) effect, we appliedphase-flipping on each image with an in-house program. The density mapswere initially reconstructed by combining 1812 individual images to aneffective resolution of 14 Å. Next, amplitude correction was appliedduring map reconstruction while new data was added. The final densitymap was reconstructed by combining images of 4348 individual particles,and the final resolution was assessed as 10.6 Å by Fourier ShellCorrelation with a cutoff of 0.5 (FIG. 26 b).

Docking of the T=1 crystal structure into the T=3 cryo-EM density mapwas first done manually with the program O (Jones, T. A. et al., Actacrystallogr. Sect. A 47, 110-119 (1991)), and then refined with theSitus software package (Chacon, P. and Wriggers, W., J Mol Biol 317,375-384 (2002)). The PORF2 monomer was treated as a rigid body duringthe initial fitting and refinement processes.

X-ray Crystallographic Structure Determination of T=1 HEV-VLP

Crystallization of the VLPs was performed according to a previouslydescribed method (Wang, C. Y. et al., Acta Crystallogr. Sect. F Struct.Biol. Cryst. Commun. 64, 318-322 (2008)). Crystals were directlyflash-frozen in liquid nitrogen and x-ray diffraction experiments wereperformed. All x-ray experiments of the HEV-VLP crystals were performedat SPrin-8 in Hyogo, Japan. Particle orientation in the unit cell wasdetermined with a self-rotation function, (Blow, D. M. et al., J MolBiol 8, 65-78 (1964)) and the particle position was determined by atranslation search with the cryo-EM structure as the model. Theasymmetric crystal unit contains one particle; as a result, 60-foldnon-crystallographic symmetry (NCS) averaging was enforced. The cryo-EMstructure (Xing, L. et al., Virology 265, 35-45 (1999)) was used toobtain the initial phases of Data I, and generated the envelope (mask)used for NCS averaging. The phases were refined by real space electrondensity averaging with icosahedral symmetry elements and solventflattening. The resolution was gradually extended to 8.3 Å(R-factor=0.21, correlation coefficient [CC]=0.92). This structure wasused for the phasing of Data II, and the phases were refined andextended to a 3.8-Å resolution (the overall R-factor and CC were 0.18and 0.97, respectively).

Sixty icosahedrally related S-subunits were treated as identical andstrict NCS constraints were applied during refinement. The data withresolution range of 20-3.8 Å was used in the refinement (Table 2 andFIG. 27). Further positional and B-factor refinement, followed by manualrevision of the mode, resulted in an R-factor of 0.242 (R_(Free)=0.245)with reasonable stereochemistry (root mean square [rms] deviations inbond lengths and bond angles were 0.010 Å and 1.68°, respectively).Because of the high NCS, the R-factor and R_(Free)-factor were almostidentical. After refinement, the stereochemistry of the structure waschecked with Procheck (Laskowski, R. A. et al., J Biomol NMR 8, 477-486(1996)): 98.1% of the nonglycine residues were within the most favoredand the additional allowed regions of the Ramachandran plot, and none ofthe residues were in the additional regions. Atomic structurerepresentations were generated using MolScript (Kraulis, P., J Appl.Crystall. 24, 946-950 (1991)) and Raser3D (Merritt, E. A. and Murphy, M.E. P., Acta Crystall. Sec D 50, 869-873 (1994)).

Results

Scanning Transmission Electron Microscopy (STEM)

The virion-sized HEV-VLP was recovered when the genotype-3 ORF2 sequencewas expressed in insect cells. This VLP projected as spherical imagewith a diameter of ˜40 nm, larger than the T=1 VLP (27 nm in diameter)(FIG. 18 a). In cryo-electron micrographs, the images of HEV-VLP aredecorated with spike-like features and are homogenous in contrast (FIG.18 b).

To determine the composition of the large HEV-VLP, we performed massmeasurements by using STEM, a technique measures the amount of electronsscattered from the objects, such as VLPs, on an EM grid. A mixture ofpurified large and small HEV-VLPs was freeze-dried onto EM grids forSTEM mass measurement. Tobacco mosaic virus (TMV) with a knownmass-to-length ratio was used as an internal standard. The HEV-VLPsappeared as spherical projections with white contrast on the dark-fieldSTEM images (FIG. 18 a). White cloud-like objects were present in thebackground, which might be the broken VLPs during sample preservation.The mean mass of large VLP and TMV in the images was measured togenerate a plot of the mean TMV mass per unit length versus mean VLPmass per particle (FIG. 18 a). A first-order fit was calculated and themass of the large HEV-VLP was determined to be 11.8 MDa (FIG. 18 d). Themass of the genotype-3 ORF2 protein, which was recovered from the largeVLP, was measured as 65.5 KDa by mass-spectrometry (FIG. 18 c).Therefore, the HEV large VLP contains 180 copies of ORF2 proteins,suggesting that the large HEV-VLP is a T=3 icosahedral particle (T=3VLP).

Three-dimensional Reconstruction of the HEV Virion-sized Particle

The cryo-EM structure of the large HEV-VLP revealed 90 protruding spikeson a complete icosahedral shell (FIG. 19 a), which is consistent withthe T=3 icosahedral symmetry and the results of the STEM massmeasurements. The VLP had an overall diameter of 410 Å and a centralcavity of 170 Å in radius as measured from the three-dimensional densitymap (FIG. 19 c). The single-layer capsid contained 180 copies of ORF2protein, which were grouped into three unique monomers according totheir geometric environments. While monomers A and B formed dimericspikes (A-B dimers) around each of the fivefold axes, two 2-fold relatedC monomers formed a spike (C-C dimer) at each of the icosahedral twofoldaxes (FIG. 19 b). The surface lattices of ORF2 proteins in large HEV T=3VLP was similar to the capsid arrangement of caliciviruses. Compared tothe A-B dimer, the morphology of the HEV C-C dimer was lesswell-defined, perhaps due to flexibility in the angle of protrudingdomain toward icosahedral shell.

The density map of the T=3 VLP displayed four discrete domains,designated from the outside inward as P, S2, S1, and N, on a section 52Å from the equatorial plane (FIG. 19 c). The density profile of the P,S2, and S1 domains displayed less variation from that observed in T=1HEV-VLP and the docking of the crystal structure of the T=1 PORF2protein to the density map of T=3 HEV-VLP showed a very good agreementbetween the two structures (FIG. 19 d). The docking positionedN-terminal tail of the PORF2 protein at the capsid inner surface alignedwell with the density linker in T=3 VLP (FIG. 19 d). The linker densityserved as a tag to connect the N domain with the icosahedral capsid,indicating the location of the N-terminal 111 amino acids of the ORF2protein in T=3 HEV-VLP.

Crystal Structure of the Genotype-1 T=1 HEV-VLP

The crystal structure of the truncated genotype-1 capsid protein (PORF2,containing residues 112-608) can be separated into three domains, S1,S2, and P, with a less resolved region covering residues 555-560. The S1domain formed by residues 118-317 folds into a classical eight-strandedβ-barrel with a jelly roll motif (FIG. 20 a), as observed in many T=3viral capsid proteins (Rossmann, M. G. and Johnson, J. E., Ann RevBiochem 58, 533-573 (1989); Harrison, S. C., Curr Opin Struct Biol 11,195-199 (2001)). Uniquely, three additional short α-helixes wereobserved in the S1 domain between strands E and F and strands G and H.The capsid shell was mainly stabilized by inter-subunit interactionsbetween the S1 domains. The folded S2 domain, consisting of residues318-451, was a twisted antiparallel β-sheet with an α-helix between theB′ and C′ strands (FIG. 20 a). The P domain, composed of residues452-606, folded into a β-barrel composed of antiparallel β-sheets,F″A″Bb″ and Ba″E″D″C″ (FIG. 20 a), and were connected with the S2 domainthrough a long proline-rich hinge (PTPSPAPSRP (SEQ ID NO:8) of residues452-461) (FIG. 20 a). Although both the S2 and P domains existed abovethe S1 domain, the protruding spikes in the HEV cryo-EM map contain onlyP domain density, which is a clear difference to those caliciviruses(FIG. 25). The PORF2 dimers have the largest buried surface area (BSA)between monomers (5,900 Å²) mainly due to between P domains (FIG. 20 b).The BSA is 3,400 Å² and 1600 Å² for the two adjacent PORF2 subunitsaround threefold axis and fivefold axis, respectively (FIG. 20 b).Moreover, the BSA of third molecule around threefold axis (9,500 Å²) ismuch wider than that around fivefold axis (4,700 ↑²).

Sequence alignment of genotype-1 PORF2 with those of genotype-3(Yamashita, T. et al., Proc Natl Acad Sci USA 106, 12986-12991 (2009))and genotype-4 (Guu, T. et al., Proc Natl Acad Sci USA 106, 12992-12997(2009)) revealed that the S1 domain is the most conserved region amongHEV genotypes, while greater divergence was seen in the N-terminalregion (FIG. 24 a). Among the solved structures, genotype-3 appearedflexible at the N-terminal end and was 11 amino acids shorter than theothers (FIG. 24 b). Because amino acids 118-129 play an important rolein bridging the N- to S1-domain in T=3 VLP and serve as dockingregisters, we used the crystal structure of genotype-1 to decipher theT=3 cryo-EM density map.

Consistent Interdimeric Interactions Between T=3 and T=1 HEV-VLPs

To understand the mechanism of ORF2 protein transition between T=1 andT=3 assemblies, we docked the T=1 decamer and hexamer into the T=3cryo-EM density map. The decamer of T=1 VLP consisted of 10 adjacentPORF2 monomers corresponding to five dimers around a fivefold axis,while the T=1 hexamer corresponded to three adjacent dimers around athreefold axis. Unlike the hexamer, the coordinates of the PORF2 decamerfitted very well with the curvature of the T=3 density map at thefivefold vertex (FIG. 21 a) and with the domain separation (FIG. 21 b).The curvature of T=3 capsid at threefold axis did not agree with thecoordinates of the PORF2 hexamer, as one of the dimers appeared stickingout of the cryo-EM density map (data not shown). Besides, theorientation of the P domain of the C-C dimer relative to its S2/S1domains was 90° different to that of the A-B dimer (FIG. 22). Thissuggests that the molecular interactions among A-B dimers in the T=3icosahedron are consistent with the dimer-dimer interactions in the T=1icosahedral assembly, while the interaction between A-B dimer and C-Cdimer is unique to the T=3 assembly.

In Vitro Reassembly of the ORF2 Protein

In order to understand the role of ORF2 decamer in T=3 VLP assembly, weanalyzed the self-assembly process of HEV-VLP in vitro. A combination ofchelating (EDTA) and reducing (DTT) agents was found to disassemble T=3VLP in a high alkaline environment (pH 10) without denaturating the ORF2protein (data not shown). Addition of 20 mM CaCl₂ into the disassemblysolution led to the association of the ORF2 dimers into star-shapedcomplexes, and no refolded VLP was found (FIG. 21 c). When we examinedthe star-shaped complexes, we found that the distance between twoopposite vertices was ˜18 nm, close to the diameter of TMV (FIG. 21 c).This size was consistent with that measured from PORF2 decamers. Thus,the star-shaped complexes resembled not only the appearance but also thesize of the ORF2 decamer (a pentamer of dimers). Although the overallBSA around threefold axis is larger than that around fivefold axis, wedid not find any complexes that could fit with PORF2 hexamer.

The in vitro disassembly-and-reassembly suggested that other factorsthan ORF2 protein contribute to T=3 VLP assembly. Considering theelectropositivity of the ORF2 N-terminal 111 amino acids, we performednucleic acid extraction from both the T=3 and the T=1 VLPs.Electrophoresis results demonstrated the presence of nucleic acids inthe T=3 extract, while the T=1 VLP extract was negative for nucleicacids (FIG. 21 d). The extracted nucleic acids were sensitive to RNasetreatment and resistant to DNase treatment, confirming that the T=3 VLPencapsidated RNA fragment during assembly while the T=1 VLP is free ofRNA fragment. This result is consistent with the VLP profiles observedfrom the cryo-electron micrographs.

Discussion

Hepatitis E virus is a human pathogen that causes acute liver failure.Like other hepatitis viruses, HEV cannot be propagated with currentlyavailable cell culture techniques. The capsid protein of genotype-3 HEVcan be expressed in insect cells as PORF2 protein, including amino acids112-608 that self-assemble into T=1 VLP, and as ORF2 protein, includingamino acids 1-608 that form T=3 VLP.

The crystal structures of PORF2 revealed three functional domains, S1,S2, and P, and the function of each domain constrained its sequenceflexibility. The S1 domain formed an icosahedral shell that served asthe base for arranging S2 and P domains; hence, the subunit surfaceshould be highly conserved among genotypes. Sequence alignment agreedvery well with this function, identifying the S1 domain as the mostconserved region among HEV genotypes (Zhai, L. et al., Virus Res 120,57-69 (2006)). The P domain serves as the putative binding site for bothneutralizing antibody and cellular receptor (He, S. et al., J Gen Virol89, 245-249 (2008)) and contains 19 divergent amino acids across fourgenotypes (FIG. 24 a). Only nine of these amino acids were exposed atthe surface of the P domain. Inspection of the binding footprint ofantibodies on the cryo-EM density map indicated that only one amino acidwas buried within the antibody-binding interface (Wang et al.,manuscript in preparation). This explains why the HEV serotype isnon-divergent despite sequence variation among HEV genotypes. The directcorrelation between sequence variability and domain functionality may benecessary for the HEV capsid to carry multiple functions and to ensureerror-free assembly. It also explains why the transition of HEV-VLP fromthe T=3 to T=1 lattice does not disturb its antigenicity, and why T=1VLP can be disassembled and reassembled in vitro to carry foreignantigenic epitopes (Niikura, M. et al., Virology 293, 273-280 (2002)) orDNA plasmids (Takamura, S. et al., Gene Ther 11, 628-635 (2004)).

The T=3 HEV-VLP has a similar morphology to that of calicivirus;however, the crystal structures of PORF2 revealed a distinctive S2domain arrangement, although the folding of the HEV S2 domain HEV issimilar to the folding of the P1 domain in caliciviruses (FIG. 25). InHEV, the P domain is located at the C-terminal end of the S2 domain,while the P2 domain of caliciviruses is inserted into the P1 domain atthe region between the A′ and B′ strands (Chen, R. et al., Proc NatlAcad Sci USA 103, 8048-8053 (2006); Prasad, B. V. V. et al., Science286, 287-290 (1999)). Furthermore, the S2 domain of HEV interactsstrongly with the S1 domain and connects to the P domain via a longproline-rich hinge, while the P1 domain in caliciviruses is a subdomainof the protrusion spike (FIG. 25). This seems to have an impact on VLPstability: the spike of the HEV C-subunits appeared weakly definedcompared to that in the A-B dimer, while the spike of the Norwalk virus(NV) C-subunit appeared rigid and similar to that in the A-B dimer inthe cryo-EM structure (Prasad, B. V. V. et al., J Mol Riol 240, 256-264(1994)). Additionally, deletion of the N-terminal positively chargedamino acids from the NV capsid protein does not induce T=1 VLP becausethe NV capsid protein only contains a short N-terminal tail of 20 aminoacids (Bertolotti-Ciarlet, A. et al., J. Virol. 76, 4044-4055 (2002)).

The HEV C-C dimer is profoundly different from the HEV A-B dimer in theorientation of the P domain relative to the S2/S1 domain (FIG. 22).Conformational difference between A-B dimer and C-C dimer has beenreported earlier on tomato bushy stunt virus (TBSV) and other T=3viruses. In TBSV, binding of RNA plays an important role todifferentiate the C-C dimer from the A-B dimer. The N-terminal arm ofthe C-C dimer is well-ordered and interacts with the RNA genome, whilethe A-B dimer is disordered and free from RNA interactions (Timmins, P.A. et al., Structure 2, 1191-1201 (1994)). In Flock House Virus, the C-Cdimeric contact acquires a flat conformation to accommodate the RNAduplex, while the A-B dimer is in a bent conformation and involves noRNA (Fischer, A. and Johnson, J. E., Nature 361, 176-179 (1993)). Thedifferent orientation observed between HEV C-C dimer and the A-B dimermay result from the difference on RNA occupancy. The A-B dimers do notinteract with RNA and have a bent conformation. As a result, the angledcontact of the S2/S1 domains accommodated the proline-rich hinge withinthe V-shaped cleft, similar to that in the T=1 VLP, thus solidifies theorientation of the P domain (FIG. 22 c). In contrast, the contact withthe RNA led the C-C dimer to a flat conformation that pushes the hingeout of the cleft. Thus the P domain in the C-C dimer is flexible andcould take a 90° rotation from the orientation in the A-B dimer (FIG. 22d).

It is suggested by molecular simulation that T=3 icosahedral capsidassembly utilizes a mechanism in which preformed aggregates ofintermediates combine in contrast to the formation of the T=1icosahedral capsid that includes the addition of predominately monomers(Ngyyen, H. D. et al., J Am Chem Soc. 131, 2606-2614 (2009)). The ORF2decamer is therefore the assembly intermediates of T=3 HEV capsid andlocated at each of the fivefold vertex. The appearance of a hexamericring at icosahedral threefold positions is the critical step in T=3capsid assembly and depends on the C-C dimer. The in vitrodisassembly-and-reassembly also indicates the involvement of extrinsicfactor than the ORF2 protein in the assembly of T=3 VLP and the C-Cdimer is in a flat conformation that is concomitant with RNA binding.The induction of C-C conformation has been reported with bacteriophageMS2, where the complete assembly of capsid requires the presence ofsynthetic RNA fragment (Stockley, P. G. et al., J Mol Biol 369, 541-552(2007)). Therefore, the interaction of RNA with the N-terminal end ofORF2 is the driving force leading the C-C dimer to the flat formationand ultimately full capsid formation through the integration of 30copies of C-C dimers with 12 copies of A-B decamers (FIG. 23).

The existence of the N-terminal 111 amino acids prevents ORF2 proteinsfrom forming T=1 VLP. The capsid of T=1 VLP encloses a central cavitywith a volume allowing maximum 55 additional residues on each copy ofPORF2 protein, if the average protein density is considered to be 1.30g/ml. The central cavity of HEV T=3 VLP is about 340 Å in diameter,which is sufficient to accommodate both the HEV genome and ORF2N-terminal domains. By characterizing the size and the N-terminalsequence of the encapsidated RNA, we found that the T=3 HEV VLPselectively encapsidated the RNA fragment that encodes the sequence ofORF2 protein (Li, manuscript in preparation). Thus, it is very possiblethat the native HEV capsid is T=3 icosahedron. There, the encapsulatedgenomic RNA may play a direct role in the assembly of HEV infectiousvirion. However, our data demonstrated here that HEV was different fromcaliciviruses in its assembly pathway, protein domain arrangement, andgenome organization, although both viruses are T=3 icosahedral particleswith dimeric spikes. Hepatitis E virus showed a high similarity to someplant viruses in its assembly pathway and its utilization of a longelectropositive N-terminal domain. Although the evolutionary origin forsuch similarities requires further investigation, our data place HEVstructure in a unique position, deviating from that of humancaliciviruses and approaching that of T=3 small plant viruses.

TABLE 2 Data collection and refinement statistics (MolecularReplacement) Crystal 1 Crystal 2 Data collection No. Crystals 1 2 Spacegroup P2₁2₁2 ₁ P2₁2₁2 ₁ Cell dimensions a, b, c (Å) 337.00, 343.00,346.00 337.00, 347.00, 354.00 α, β, γ (°) α = β = γ = 90° α = β = γ =90° Resolution (Å) 70.0-8.30 (8.60-8.30) 70.0-3.80 (3.94-3.80) R_(sym)or R_(merge) 0.136 (0.500) 0.136 (0.626) I/σI 8.3 (2.2) 6.3 (1.0)Completeness (%) 87.6 (89.4) 68.4 (24.8) Redundancy 3.0 (3.0) 3.4 (1.4)Refinement Resolution (Å) 20.00-3.80 No. reflections 275, 008R_(work)/R_(free) 0.242/0.245 No. atoms Protein 3.659 Ligand/ion 0 Water0 B-factors Protein 140.7 Ligand/ion Water R.m.s deviations Bond lengths(Å) 0.010 Bond angles (°) 1.7

Example 5 Engineered Nucleopeptide Capsid of Acute Hepatitis E VirusPossesses Functional Modularity as Oral Vaccines

Hepatitis E virus (HEV) is a water-borne viral agent and primarilytransmitted via fecal-and-oral route thus resistant to low pH anddigestive enzymes associated with stomach and digestive tracts. Theinfection of HEV causes acute hepatitis in human with a mortality rateup to 30% in pregnant women (Naik, S. R. et al., Bull World HealthOrgan, 70 (5), 597-604 (1992)). Currently, there are 1,600 genomicsequences of HEV available at the International Nucleotide SequenceDatabase Collaboration (Khuroo, M. S. & Khuroo, M. S., Curr Opin InfectDis, 21, 539-543 (2008)), which are grouped into four genotypes (Jameel,S., Expert Rev Mol Med, 1999, 1-16 (1999)). Notably, only a single knownserotype is recognized, suggesting that the immuno-dominant domain ofHEV is highly conserved. The major capsid protein ORF2, encoded at thesecond open reading frame, is reported most immunogenic and isresponsible for the induction a protective humoral immune response((Li,T. et al., Vaccine, 22, 370-377 (2004); Purdy, M. A. et al., J MedVirol, 41 (1), 90-94 (1993); Riddell, M. A., Li, F., & Anderson, D. A.,J Virol, 74 (17), 8011-8017 (2000)). Recombinant HEV capsid protein(PORF2) covering amino acids 112-608 can self-assemble into virus likeparticle when expressed in insect cells (Li, T. C. et al., J Virol, 71(10), 7207-7213 (1997)). Although HEV virion is a non-envelopedicosahedral particle with a diameter of 320-340 Å as shown byimmuno-electron microscopy (Balayan, M., Andiaparidze, A., Savinskaya,S., & et. al., Intervirology, 20, 23-31 (1983)), self assembledvirus-like particles have a diameter of 270 Å determined from its threedimensional reconstruction. The HEV-VLPs possesses HEV nativeimmunogenicity and is capable of inducing anti-HEV systemic and mucosalantibodies when orally administered to non-human primates (Li, T. etal., Vaccine, 22, 370-377 (2004)). Although the HEV-VLP was determinedas T=1 icosahedral particle composed of 60 copies of PORF2 proteins,detailed structural information about amino acids arrangement is stillnecessary to better utilize HEV VLP either as derict mucosal vaccineagainst hepatitis E or as delivery carrier to activate immune responseat mucosal surface.

To acquire in-depth the structural insight of HEV capsid configuration,we determined the crystal structure of HEV-VLP derived from genotype 1capsid protein to 3.8 Å resolution by molecular replacement techniques,as initially phased by with a 30 Å cryo-EM density map (Xing, L. et al.,Virology, 265 (1), 35-45 (1999)) (Table S1). As seen in the cryo-EMstructure, the HEV-VLP contains 30 large protruding spikes at each oficosahedral twofold axis (FIG. 28 a). The structure of the capsidprotein can be separated into three domains, S1 domain, S2 domain, and Pdomain, with a less resolved region covering residues 555-560 (FIG. 28b). The S1 domain, formed by residues 118-317, folds into a classicaleight-stranded (3-barrel with a jelly roll motif, as seen in many T=3viral capsid proteins (Rossmann, M. G. & Johnson, J. E., Ann RevBiochem, 58, 533-573 (1989); Harrison, S. C., Curr Opin Struct Biol, 11,195-199 (2001)). These eight β strands, denoted B-I, are organized intotwo antiparallel β-sheets, with two α-helices between the strands C andD and strands E and F (FIG. 28 c). Uniquely, three additional shortα-helixes were observed in the S1 domain between the strands E and F andstrands G and H. The capsid shell appears to be mainly stabilized by theinter-subunit interactions between the S1 domains. The folding of S2domain, consisting of residues 318-451, is a twisted antiparallelβ-sheet and a α-helix between B′ and C′ strands (FIG. 28 c). S2 domainis raised mainly around the 3-fold axis (FIG. 28 c), and interactsstrongly with the underneath S1 domain, with a buried surface area (BSA)of ˜3,200 Å2 (FIG. 29 a). Both the S2 and the P domains exist above theS1 domain, however, the protruding spikes in the cryo-EM map includesolely the P domain density. The P domain, composed of residue 452-606,folds into a β-barrel composed of antiparallel β-sheets, F″A″Bb″ andBa″E″D″C″ (FIG. 28 c) and connects with the S2 domain through a longproline-rich hinge (PTPSPAPSRP (SEQ ID NO:8) of residues 452-461) (FIG.28 c). This HEV capsid protein demonstrates a clearly difference in thespatial domain organization and the sequence of the three domains and astrong similarity in the folding of each individual domain to the capsidproteins of caliciviruses reported to date (Prasad, B.V.V. et al.,Science, 286 (5438), 287-290 (1999); Chen, R. et al., Proc Natl Acad SciUSA, 103, 8048-8053 (2006)).

The subunit interface at each of the twofold axis demonstrates thelargest BSA on monomers (5,900 Å²) mainly due to the contacts at Pdomains (FIG. 29 a). This interface buries a group of non-polar aminoacids involves strong hydrophobic interactions. The side chain of aminoacids of Val 470, Trp472, Val 503, Val598 and Val600 from one subunit isembraced by the hydrophobic surface patch from its dimeric partner (FIG.29 b). This hydrophobic contact is protected by a rim of polarinteractions on each site, including a hydrogen-bond made betweenresidue Trp548 of one subunit and residue Arg542 from its twofoldpartner. Thus the PORF2 dimer is stable in solution and the dimerizationwas reported to be consistent with the presence of amino acids 585-610(Li, X., Zafrullah et al., J Biomed Biotechnol, 1 (3), 122-128 (2001)).Further mutagenesis analysis confirms that the dimeric interactions isin association with the presence of six hydrophobic amino acidsresidues, Ala597, Val598, Ala599, Val600, Lue601, and Ala602 (Li, S. etal., Vaccine, 23 (22), 2893-2901 (2005)). In the crystal structure,these residues located at P domain either at the dimeric interface orwithin the hydrophobic core of the β-barrel. In addition, the S1 domainsarranges hydrophobic residues at the dimeric interface, particular theβB-strand and α1-helix, as an additional force to stabilize HEV dimer.Therefore, the capsid protein dimers are likely the building block inthe assembly of HEV-VLP and insertion at these interface region wouldinhibit the formation of HEV-VLP.

To assemble ORF2 dimers into an icosahedral shell, 30 copy of PORF2dimers have to be positioned following the strict fivefold and threefoldsymmetry where large surface area of each dimer is buried in during theassembly. As a result, the surface of S1 domain is only accessible atregion around fivefold axis thus the fivefold connection involves theresidues solely from S1 domain with 4,700 Å2 buried surface area. Thefivefold apex is surrounded by a ring of five Try288 residues with theiraromatic side chain pointing towards the center (FIG. 29 c). Theconnection around threefold axis invovles both S1 and S2 domains with9,500 Å2 buried surface area (FIG. 29 a). A density voided cavity wasobserved at threefold axis where residues Gln421 from S2 domain andAsn255 from S1 domain were observed to occupy the outermost andinnermost amino acids, respectively. The wall of this hollow is highlynegatively charged and contains three amino acids Glu269, Glu270 andGlu417 (FIG. 29 d). At current resolution, we are unable to detect anyelectron density for metal ion in the structure. However, HEV-VLP can bedisassembled in an environment containing both EDTA and DTT, whilereassembled by addition of calcium ion. The calcium-ion dependentstability suggests the existence of divalent ion in the capsid, althoughthe exact location of the divalent ion requires further systemicanalysis, for example derivatizing crystals with samarium. As the“energy landscape” of macromolecular assembly has been suggested to takestepwise process, in which the larger interface is most likely conservedin evolution (Levy, E. D. et al., Nature, 453, 1262-1265 (2008)). TheBSA of dimer-dimer contact suggests a possible assembly route of PORF2dimer through hexamer (trimer of dimers) around threefold axis becauseit conducts large BSA compared to the decamer (pentamer of dimers)around fivefold axis.

Sequence alignment of capsid protein from the four genotypes of HEVrevealed that the S1 domain is the most conserved region, while S2domain is hyper variable among genotypes (Zhai, L., Dai, X., & Meng, J.,Virus Res, 120, 57-69 (2006)). The heavy involvement of S1 domain insealing the VLP capsid with low solvent accessibility may keep thisdomain region of protein sequence more conserved among genotypes. Thereare 9 divergent residues in S2 domain and all of them locate at thesolvent-exposing surface, however, none of the known HEV antigenicepitope was located at the S2 domain.

The P domain is highly exposed and serves as the binding sites forneutralizing antibody (He, S. et al., J Gen Virol, 89 (1), 245-249(2008)). The neutralization epitope of HEV is reported to beconformational (Schofield, D. J. et al., Vaccine, 22 (2), 257-267(2003); Meng, J. et al., Virology, 288, 203-211 (2001) and the peptideincluding residues 459-607 of the capsid protein is then identified asthe minimal structure to induce anti-HEV neutralizing antibody (Zhou, Y.H., Purcell, R., & Emerson, S., Vaccine, 23, 3157-3165. (2005)). Thisregion coincides with the P-domain revealed in our structure and doesnot involved in the formation of icosahedral shell. The results ofsequence alignment revealed 19 divergent residues at P domain and 9 ofthem on the VLP surface (FIG. 30 a). Strikingly, only one of themlocates within the antibody binding interface, which is furtherconfirmed by cryo-EM structure of antibody-conjugated HEV-VLP(manuscript in preparation). There has been only one HEV serotypereported so far, regardless the fact of four existing genotypes. Thedivergent amino acids of four genotypes are located exterior on thesurface of the anigenic domain. Modularization of PORF2 protein byassigning assembly function to the S1 domain and immunogenicity to the Pdomain essures HEV-VLP preserving its native antigenic conformation eventhe VLP is smaller than the native virion. Alternatively, makingmutation or engineering of P domain will not affect the assembly ofHEV-VLP.

Four insertion sites were previously selected after residues A179, R366,A507, and R542 according to the conveniece of restriction enzyme. Thesefusion proteins failed in VLPs production (Niikura, M. et al., Virology,293 (2), 273-280 (2002)) due to the wrong spatial positions of theseresidues. The residue A179 is located in the S1 domain in the middle ofα1 helix. This helix is necessary for the integrity of S1 domain and theinteraction with its twofold related neighboring subunit. R366 islocated in the S2 domain and favors electrostatic interaction withresidue E386 from its threefold related neighboring subunit. Althoughlocated at P domains, the side chain of R542 is within the dimericinterface and guides the hydrophobic interaction of two monomers.Deletion of R542 may misalign the orientation two P domains that weakenthe dimeric interaction of PORF2 protein. The role of residue A507 inthe P-domain plays an important role in maintaining P domain orientationby fixing the angle to the long proline-rich hinge. None of the fourresidues are exposed on the surface of VLP, although some of them are atthe surface of individual PORF2 subunits (FIG. 30 b). Therefore,insertion of a foreign sequence at these sites induces no interferenceto the expression of individual protein, but does bring in hindrance tothe assembly of HEV VLPs. The crystal structure revealed that theC-terminus is exposed on the surface of VLP and is suitable to tetherbulky foreign antigenic sequences, as it showed in the previous report(Niikura, M. et al., Virology, 293 (2), 273-280 (2002)). To create amucosal vaccine against HIV infection, a HIV immunogenic epitope P18 wasinserted to the C-termini of PORF2 protein.

The infection of human immunodeficiency virus (HIV) is a major healthproblem that results in an estimated 2.7 million new infections and 2million deaths in 2007. Mucosal vaccine is particular rubust intargeting the initial infection and replication of HIV because themajority of primary infections of HIV occur at mucosal site (Miller, C.L., McGhee, J. R., & Gardner, M. B., Lab. Invest., 68, 129-145 (1993)).HIV P18 peptide (RIQRGPGRAFVTIGK; SEQ ID NO: 9) is a specific HIVpeptide located at the third variable domain of HIV-1 envelopeglycoprotein. This domain contains a T helper (Th) and the principalHIV-1 neutralization epitopes. The P18 peptide is immunodominant andable to stimulate HIV-specific CTL response (Achour, A. et al., J Virol,70, 6741-6750 (1996)). Fusion of P18 peptide to the C-terminal end ofPORF2 protein did not affected the assembly of HEV-VLP. As a result, theinserted P18 epitope is positioned adjacent to HEV immunodominantepitope and expected to be accessible to the host immune system. EachHEV-VLP will carry 60 copies of P18 epitope, with spacing of 56 Å due toits icosahedral symmetry. Mice were then orally immunized three times at1 week interval with 50 mg of purified chimeric P18-VLP in the absenceof adjuvant. HIV Env-specific IgG antibodies were detected in sera andin intestinal fluid with a level higher than those in mice that hadreceived synthetic P18 peptide (FIG. 30 c). Moreover, specific IgAantibodies to HIV env-protein were detected from the mouse intestinalfluid, while the level of IgA antibodies in sera showed no difference tothat detected in peptide-immunized mice (FIG. 30 c). The control miceimmunized with wild type HEV-VLP developed no antibodies specific to thesynthetic P18 antigen. This suggests that the chimeric P18-VLP iscapable of inducing both systemic and mucosal immunity in mice.

The chimeric P18-VLP, like the HEV-VLP, contains a cluster fourpositively charged amino acids in the interior surface of capsid aroundthe icosahedral twofold axis (FIG. 31 b), forming a local environmentfavorite nucleotide interactions. When DNA plasmid was added in thereassembly buffer, the reassembled chimeric VLP can incorporated withthe DNA plasmid. We therefore produced a chimeric VLP bearing HIV P18epitope on the surface and an encapsulated DNA vaccine expressing entireGag protein (P-P18/NGag capsule) and evaluated its immunogenicityexperimentally with mouse. After orally immunizing four times at 1 weekinterval of P-P18/N-Gag capsule, the mice developed both HIV specificIgG and IgA antibodies in serum and in intestinal fluid with a levelhigher than those in mice that had received either synthetic P18 peptideor naked DNA plasmid. Strikingly, cytotoxic T lymphocyte (CTL) responseswere detected in response to P18 epitope (FIG. 31 c) and to HIV gagprotein (FIG. 31 d) in the spleen, mesenteric lymph nodes and Peyer'spatch cells from the mice orally administrated with P-P18/N-Gag capsule.The specific CTL responses did not observed in the same tissue cellsfrom the control animals fed with either synthetic P18 peptide or nakedDNA vaccine expressing gag protein developed. This result demonstratesthat gene on DNA plasmid could be expressed in epithalial cells in thesmall intestine after delivery by HEV-VLPs.

The oral delivery of an HIV DNA vaccine for induction of mucosal immuneresponses is challenged by the difficulties to protect plasmid DNA fromgastric environment, although the efficacy can be improved byencapsulating DNA in poly(lactide-coglycolide) microparticles (Kaneko,H. et al., Virology, 267, 8-16 (2000)). HEV-VLP, derived from an orallytransmissible virus, is composed of 60 copies of PORF2 protein thatmodularized its three domains for different functions, such as antigenicpresenting and VLP assembly. Such structural modularity of capsidprotein allows HEV-VLP to retain the transmission and immunogenicproperties of the native HEV virion, thus to be incorporated intoHEV-permissive epithelial cells in the small intestine. Furthermore,these results demonstrated that our HEV-based P-P18/NGag capsule wascapable of taking the advantages of HEV-VLP in going through the mucosalsystem by not only delivering the antigen to mucosal surface, but alsonotably inducing the humoral and the cellular mucosal, as well assystemic, immune responses. Since the HEV-VLP itself is under clinicaltrials as an HEV vaccine in humans (Shrestha, M. P. et al., N Engl JMed, 356 (9895-903) (2007)), this delivery system of HEV-VLP provides afacile novel tool of oral vaccine delivery as a non-replicating entitythat can induce mucosal immunity without any adjuvant.

Example 6 HEV-VLP Encapsulating a DNA Vaccine

This invention of vaccine platform will not simply deliver anencapsidated a plasmid DNA but will also attach a peptide epitope to thesurface of the virus-like particles (VLP) as both adjuvant and boosterto maximize the efficacy. This should make feasible the induction ofcellular immunity, both helper T cells and, importantly for viralinfection, MHC class I-restricted cytotoxic T lymphocytes. This attemptis considered to be potent than the general VLP delivery system when theVLP is used to carry one type of antigen, either peptide or DNA, becausethe attached antigen functions as both adjuvant and booster to enhancethe efficacy of the DNA vaccine. The whole production does not requirethe handling of potentially deadly influenza virus and significantlyshorten production time, thus composed a feature paramount with anongoing pandemic. The encapsidation of DNA vaccine is done entirely invitro; therefore M2e conjugated HEV VLP can be produced in advance as anenvelope to enclose the DNA plasmid containing RNA segments of thepotential pandemic virus that was generated through reverse genetics.

The natural route of infection for HEV is the fecal-oral route, and thestructure of HEV-VLPs enables it to survive the low pH of the stomach inorder to pass through to the small intestine where infection of theintact virus normally occurs. Thus as a vaccine delivery system, it isable to induce mucosal immunity and even advantageous compared to, forexample, certain cholera vaccines which require pre-administration of abuffer. An effective vaccine must be capable of being readilymanufactured. The HEV-VLP can be produced from standard cultivationmethodologies with a yield of purified HEV-VLPs in the range of 50-100μg/ml, nearly 100 times greater when compared to other VLPs. Theproduced capsid is stable at room temperature and can be transported inthe absence of cold facility, a critical feature for its potency,particular in the remote low-resource regions. Mucosal surface builds upthe front line of defense in human against the entry of infectiousmicroorganisms. This is particular true for the infection of humanimmunodeficiency virus (HIV) because the majority of HIV primaryinfection takes place at mucosa. In addition, injected vaccines requirethe use of sharps (needles) or injectors, both of which have thedisadvantages of causing pain (which results in a number of peopleavoiding immunization), and of creating a biohazard. Because trainedpersonnel are required to administer such vaccines, administration of aninjected vaccine is likewise more costly than for an orally administeredvaccine, for example. However, the development of mucosal vaccine reliesheavily on the efficiency of antigen delivery system, which has to bestrong enough to protect antigen against the harsh environment, such asproteolytic enzymes in human digestive tract, and targeting the mucosalsurface.

HEV-VLP is able to activate mucosal immunity: Human hepatitis E virus(HEV) is a water-borne non-enveloped virus that transmitted primarilythrough the contaminated water. The Hepatitis E virus like particle(HEV-VLP) is composed of the truncated ORF2 protein (PORD2) derived fromthe capsid protein. When expressed in insect cells, the PORF2 proteinself-assembles into T=1 icosahedral particle with a high yield(milligram quantities at laboratory scale). We then analyzed itsstructure with both cryo-electron microscopy and image reconstructionand recently by X-ray crystallography. The VLP consists of 60 copies ofPORF2 protein and contains no nucleotide, thus unable to replicate inhost cells. Although it is smaller than the native virion (27 nm vs 34nm), this VLP demonstrated similar morphological features as the nativeHEV virion: showing protruding spikes on the surface of the icosahedralshell. Additionally, it also retains HEV native antigenicity and capableof inducing antibodies in experimental animals and protection innon-human primate. Taking the advantage of HEV natural infection route,the detection of IgA antibody in both mice and cynomolgus monkeys afteroral vaccination demonstrates that HEV-VLP is capable of elicitingmucosal immunity.

HEV-VLP is able to transfer genes in vitro and in vivo: The structure ofHEV-VLP revealed that the inner VLP inner surface contains a dominantpositively-charged patch at each of icosahedral two-fold axis,indicating that the VLP retains the intrinsic capacity of encapsidationnucleic acids. Thus this HEV-VLP is able to encapsulate DNA plasmid invitro during VLP refolding and transfer GFP-gene into the culture cellsderived from mice, rabbit, monkey and human. Combination of EGTA and DDTcan disassemble the HEV-VLP into free dimers and the disrupted VLPs canbe reassembled when calcium ions are supplemented. No significantmorphological changes were observed after negative staining Toincorporate DNA vaccine, plasmid DNA encoding target gene was mixed withthe disrupted VLP before refolding. The plasmid-encapsidate VLP is thenseparated from the empty VLP by CsCl density gradient (FIG. 32). Thefluorescence of GFP-expressing cells was observed under a fluorescencemicroscope. Although the percentages of GFP transfected cells were notso high (11.2% of NIH3T3 cells, 19.6% of RK-13 cells, 21.0% of COS-7cells, and 20.1% of HepG2 cells), all of the cell lines tested in thestudy showed positive reaction, in contrast to the cells that wereincubated with plasmid DNA alone or intact HEV-VLP in the presence ofplasmid DNA. To test whether HEV-VLP could induce gene transduction invivo, we reassembled HEV-VLP so it encapsulated plasmid DNA expressingHIV env gp120 of the NL432 strain (pJWNL432). Mice that orally receivedthis VLP were killed two days after immunization and the expression ofHIV env protein was found in epithelial cells of the small intestine byimmunohistochemistry. These data demonstrated the ability of HEV-VLP asgene carrier for mucosal delivery.

HEV-VLP delivery is able to induce specific mucosal immune responses inmice: Mice were orally and subcutaneously immunized with either HEV-VLPcontaining pJWNL432 or naked pJWNL432 plasmid four times at 1-weekintervals. Sample was collected from mice serum and fecal at differenttime points after immunization and the level of antibody induction wasexamined with ELISA, against HEV-VLP, synthetic HIV P18 peptide, andCV-1 cells infected with recombinant Sendai virus expressing HIV gp120protein of NL432 strain. The serum levels of HIV env-specific IgGantibodies detected in sera from subcutaneously and orally immunizedmice are eventually the same, while no IgG was detected in any of thefecal samples. However, the level of IgG are significantly higher insera of the mice received DNA-loaded VLP than that had received nakedDNA (P<0.05 at 12 wpi) (FIG. 33). Here, the statistical analysis wasperformed using Mann-Whitney's U-test and Kruskal-Wallis test.

Importantly, specific IgA against HIV env is only detected at highlevels in the sera of mice that had been immunized orally withDNA-loaded VLP but not in the sera of mice that had been immunizedsubcutaneously (P<0.05 at 12 wpi). Such specific IgA was only detectedin fecal extracts of the mice that had orally received DNA-loaded VLPs(FIG. 33). These data indicate that HEV-VLP is effectively deliver DNAvaccine to mucosal surface.

HEV-VLP delivery is able to elicit specific CTL responses in mice: Oneof the significant advantages of DNA vaccine is to stimulate bothantibody and T-cell arms of the immune system inducing those that arespecialized to kill viruses via cytotoxic or killer T-cells. To examwhether HEV-VLP delivery induces cellular responses; we investigatedcytotoxicity by 51Cr-release assay on the effector cells that wascollected from spleen, mesenteric lymph nodes (MLN) and Payer's patch(PP) cells at five weeks after first immunization. The P18 peptide is adominant HIV env CTL and Th cell epitope in BALB/c mice and isrestricted to the H-2D^(d) allele Inhibition of these effector cells byeither anti-CD8 or anti-H-2D^(d) monoclonal antibody was alsoinvestigated. The results revealed that the mice can develop HIV-envepitope-specific CTL responses in spleen, MLN, and PP after receivingplasmid-loaded HEV-VLP, which the mice received naked DNA plasmid didnot develop such specific CTL responses (FIG. 34). The function of theseeffectors cells were inhibited by anti-CD8 and anti-H-2D^(d) monoclonalantibodies. Therefore, oral administration of mice with HEV-VLPencapsidated HIV env DNA vaccine elicited CD8+ and MHC classI-restricted CTL responses both locally and systemically.

Both HIV antigenic epitope P18 and DNA vaccine were delivered usinghuman hepatitis E virus capsid particles. P18 epitope is located in thethird variables domain (V3 loop) of the HIV envelope glycoprotein andcontains a T helper and the principle HIV neutralization epitopes.Administration of P18 is able to induce anti-HIV CTL responses thatappear to play an important role in the control of HIV infection bykilling the infected cells. The HIV DNA vaccine is plasmid encoding HIVsoluble glycoprotein gp120. The expressed gp120 protein can be selectedfor presentation at the surface of infected cells by association withmajor histocompatibility complex class II (MHC II) molecules, thusactivate CTL responses. It is known that HIV-specific CTL responses havea critical role in controlling HIV replication because CD8+ T lymphocyteresponses emerge during acute infection coincident with initial controlof primary viremia. With the structural information achieved lately, weare able to modify HEV capsid protein so as to let such capsid deliverboth P18 epitope and gp120 gene simultaneously to the mucosal surfacethrough oral administration. Therefore, the proposed system canstimulate induction of systemic and mucosal antibodies as well ascellular CTL response against HIV infection. HEV-VLP, a highly orderedclosure capsid but lacking viral genomic material, retains the overallstructure of viral capsid and showed great advantage in delivery ofsmall molecule, antigenic epitopes, and therapeutic gene. Inparticularly, HEV capsid, as derived from gastroenteric transmittingagents, inherits the natural transmission pathway to target mucosalantigen-presenting cell through oral vaccination. This technology shouldinduce not only antibodies and cellular immunity, but also mucosalimmunity (at the site of infection), to play effective role in 1) theinduction of anti-HIV antibodies to reduce the virus inoculum, as wellas in 2) the activation of cellular responses to facilitate theclearance of HIV-infected CD4 cell.

Example 7 Various Chimeric HEV-VLP Constructs

Various heterologous peptides were inserted into the portion of HEV ORF2 within a pre-selected region of residues 483-490, residues 530-535,residues 554-561, residues 573-577, residues 582-593, or residues601-613 of the HEV ORF 2 protein. The heterologous epitopes can be usedin these constructs include, but are not limited to, HIV-V3, flu-M2, HSVand reo (diarrhea viruses). Various constructs have been made,including: (1) a HEV-VLP with HIV V3 peptide inserted into the region ofresidues 483-490; (2) a HEV-VLP with HIV V3 inserted into the region ofresidues 530-535; (3) a HEV-VLP with HIV V3 peptide inserted into theregion of residues 554-561; (4) a HEV-VLP with HIV V3 peptide insertedinto the region of residues 582-593; and (5) a HEV-VLP with HIV V3peptide, or a flu-M2 peptide, or a herpes simplex virus peptide,inserted into the region of residues 601-613.

Example 8 Spatial Configuration of Hepatitis E Virus Antigenic Domain

Hepatitis E virus (HEV) is a human pathogen that causes acute hepatitis.When an HEV capsid protein containing a 52-amino acid deletion at theC-terminus and a-111 amino acid deletion at the N-terminus is expressedin insect cells, the recombinant HEV capsid protein can self-assembleinto a T=1 virus-like particle (VLP) that retains the antigenicity ofthe native HEV virion. In this study, the inventors used cryo-electronmicroscopy and image reconstruction to show that anti-HEV monoclonalantibodies bind to the protruding domain of the capsid protein at thelateral side of the spikes. Molecular docking of the HEV VLP crystalstructure revealed that the Fab224 covered three PORF2 surface loops atthe top part of the spike. The inventors also determined the structureof a chimeric HEV VLP and located the inserted B-cell tag, an epitope of11 amino acids coupled to the C-terminal end of the recombinant ORF2protein. The binding site of Fab224 appeared distinct from the locationof the inserted B-cell tag, suggesting that the chimeric VLP couldelicit immunity against both HEV and an inserted foreign epitope.Therefore, the T=1 HEV VLP is a novel delivery system for displayingforeign epitopes at the VLP surface in order to induce antibodiesagainst both HEV and the inserted epitope.

Introduction

Hepatitis E virus (HEV) is a causative agent of acute hepatitis inhumans and is primarily transmitted via the fecal-oral route. HEV isthus resistant to the low pH and digestive enzymes associated with thestomach and gastrointestinal tract. HEV regularly causes epidemics inmany tropical and subtropical countries. In India, 101 outbreaks wereconfirmed by serological analysis in the state of Maharashtra in thelast 5 years (Deshmukh et al., 2007, Vaccine 25:4350-4360), and inIndia, the lifetime risk of HEV infection exceeds 60% (Worm andWirnsberger, 2004, Drugs 64:1517-1531). Sporadic cases have also beenreported in HEV-endemic regions, as well as in non-endemic areas.Although some of these cases were associated with travel, many casesinvolved patients without history of travel to HEV endemic regions.Accumulating evidence suggests that sporadic infection occurs through azoonotic route and is not limited to developing countries.Seroprevalence suggests hepatitis E infection may also be prevalent inthe United States (Minuk et al., 2007, CAn J Gastroenterol. 21:439-442),France (Bendall et al., 2008. J Med Virol 80:95-101), and Japan(Mushahwar, 2008, J Med Virol 80:646-658). The overall mortality rate ofHEV infection during an outbreak generally ranges from 1 to 15% and thehighest mortality occurs in pregnant women, with fatality rates of up to30% (Naik et al., 1992, Bull World Health Organ 70:597-604).

The HEV virion is composed of a 7.2 kB single-stranded RNA molecule anda 32-34 nm icosahedral capsid. The HEV genome contains three openreading frames (ORFs). The capsid protein, encoded by the second openreading frame (ORF2) located at the 3′ terminus of the genome, comprises660 amino acids and is responsible for most capsid-related functions,such as assembly, host interaction, and immunogenicity. Recombinant ORF2proteins can induce antibodies that block HEV infection in non-humanprimates (Li et al., 2004, Vaccine 22:370-377; Tsarev et al., 1997,Vaccine 15:1834-1838). Four major antigenic domains were predicted to belocated within the C-terminal 268 amino acids of ORF2 protein, onedomain was experimentally identified as a neutralization epitope in theSar-55 ORF2 capsid protein (Schofield, et al., 2000, J Virol74:5548-5555; Schofield et al., 2003, Vaccine 22:257-267). However, theminimal peptide needed to induce anti-HEV neutralizing antibodiescontains residues 459-607 of the ORF2 protein (Zhou et al., 2005,Vaccine 23:3157-3165), which is much longer than a linear antigenicepitope, suggesting that the neutralization epitope is conformational.Therefore, the detailed structure of HEV capsid protein is required inorder to understand the organization of HEV epitopes.

Currently, there are 1,600 HEV genomic sequences available through theInternational Nucleotide Sequence Database Collaboration. They areclassified into four genotypes, which vary by geographic distributionand host range (Khuroo et al., 2008, Curr Opin Infect Dis 21:539-543).In contrast, only a single serotype has been identified, suggesting thatthe immuno-dominant domain of HEV is highly conserved among genotypes.Antibodies from any one of the four genotypes cross-react with thecapsid protein of genotype-1 (Emerson et al., 2006, J Gen Virol87:697-704).

Like other hepatitis viruses, HEV does not propagate well in currentlyavailable cell culture systems. Hepatitis E preventive strategies so farrely on the use of ORF2-derived recombinant protein (Maloney et al.,2005, Vaccine 23:1870-1874). When expressed in insect cells, truncatedrecombinant ORF2 protein (PORF2) with 52 residues deleted from theC-terminus and 111 residues deleted from the N-terminus, self-assemblesinto virus-like particles (VLPs) (Li et al., 1997, J Virol71:7207-7213). The inventors' previous structural analysis ofrecombinant HEV VLP by cryo-electron microscopy (cryo-EM) provided thefirst understanding of the quaternary arrangement of PORF2.

The essential assembly element of PORF2 protein contained amino acids125-600 (Li et al., 2005, J. Virol. 79:12999-13006) and thereconstructed VLP displayed a T=1 icosahedral particle composed of 60copies of truncated PORF2 (Xing et al., 1999, Virology 265:35-45).Recently, crystal structures were reported for genotype-1 T=1 VLP (Xinget al., 2010, J Biol Chem 285:33175-33183), genotype-3 T=1 VLP(Yamashita et al., 2009, Proc Natl Acad Sci USA 106:12986-12991), andgenotype-4 T=1 VLP (Guu et al., 2009, Proc Natl Acad Sci USA106:12992-12997), revealing that PORF2 is composed of three domains:S-domain, M-domain and P domain. The T=1 icosahedral shell is composedof 60 copies of S-domains, while the M-domain binds tightly to theS-domain and interacts with two threefold-related M-domains to form asurface plateau at each of the three-fold axes. Two P domains aretightly associated as a dimeric-spike that protrudes from each of theicosahedral twofold axes. As a result, on a low resolution cryo-EMdensity map, the HEV T=1 VLP appears as an icosahedral particle with 30spikes (Xing et al., 1999, supra).

Although these VLPs are smaller (270 Å in diameter) than the native HEVvirion (320-340 Å), oral administration of HEV VLPs to experimentalanimals can induce anti-HEV antibodies that bind to native HEV (Li etal., 2001, Vaccine 19:3476-3484). When a B-cell tag of 11 amino acids onglycoprotein D of herpes simplex virus was covalently coupled to theC-terminal end of PORF2 (after residue 608), the fusion protein retainedthe ability of PORF2 to assembly and form chimeric T=1 icosahedral VLPthat were capable of eliciting systemic and mucosal antibodies againstboth HEV capsid protein and the attached B-cell tag (Niikura et al.,2002, Virology 293:273-280). Therefore, the HEV T=1 VLP is a potentialcarrier for delivering not only HEV antigen but also foreign antigens oranti-viral drugs to the host immune system. However, rational design ofHEV-based delivery vectors requires detailed information on HEV VLPstructure as well as HEV immunodominant domains.

In this study, the inventors identified antigenic structures usingcryo-EM and three-dimensional reconstruction. Our results indicate thatthe binding footprint of a neutralizing antibody covers the lateral sideof the P domain, while a B-cell tag at the C-terminus does not alter theassembly of T=1 HEV VLP.

Materials and Methods

Production and Purification of Anti-HEV Monoclonal Antibody Mab224

Eight-week-old female BALB/c mice were immunized at 0 and 4 weeks byintraperitoneal inoculation with HEV VLPs (100 ug/ml). Four weeks later,a final boost containing an equal volume of antigen was administered.Three days after the final boost, mouse spleen cells were fused withP3U1 mouse myeloma cells using polyethylene glycol 1500 (50% [w/v])(Boehringer, Mannheim, Germany) essentially as described by Adler andFaine (Adler and Faine, 1983, Pathology 15:247-250). Supernatants frommicroplate wells positive for hybridoma growth were screened by ELISAusing recombinant HEV VLPs as the antigen. Hybridomas that secreteantibodies specific for HEV were subcloned three times by limitingdilution, after which they were considered to be monoclonal. Antibodiesin the supernatants were isotyped using the Mouse Monoclonal AntibodyIsotyping kit (Amersham, Little Chalfont, Buckinghamshire, U.K.) inaccordance with the manufacturer's protocol. Hybridomas were grown inbulk in stationary flasks (Nunc, Roskilde, Denmark) using RPMI-1640 with15% FCS. Antibodies were purified from cell supernatants using HiTrapprotein G affinity columns (Pharmacia Biotech AB, Uppsala, Sweden) andstored at −80° C. Among all of the antibodies that were generated,Mab224, an immunoglobulin G1 (IgG1) isotype was chosen for structuralanalysis.

Preparation of Fab224 Fragments

Isolated Fab224 fragments were prepared from purified mouse monoclonalantibodies by papain cleavage. A reducing L-cysteine buffer was used toactivate the papain, and MAb224 was mixed with papain at a molar ratioof 100:1. The mixture was incubated overnight at 30° C. The reaction wasstopped by the addition of iodocetamide, and the product was analyzed bySDS-PAGE. The Fab224 fragments were purified using a 5-ml pre-packedProtein-A chromatography column (Pierce Protein Research) according tothe manufacturer's instructions. The Fc fragments and un-cleaved MAbs224were trapped in the column due to their affinity for protein-A, whilethe Fab224 fragments were collected in the flow-through fraction.

Production and Purification of Anti-HEV Fab4

The Fab4 was prepared by phage display and purified according to theprotocol described previously (Schofield, et al., 2000, J Virol74:5548-5555). Briefly, chimpanzee 1441 was infected with HEV strainSAR-55. Bone marrow was aspirated from the iliac crest of this animaland antibody κ-chain gene and γ1-chain gene were amplified and clonedinto pComb3H phage display vector and pGEM-T cloning vector (Promega)respectively, and transformed into E coli XL-1 Blue. The bacteria werethen amplified and infected with helper phage VCS M13 at multiplicity ofinfection of 50 to produce a library displayed on surfaces of phageparticles. Phage was panned on SAR-55 ORF2-coated ELISA wells; fourrounds of panning were performed. After amplification of the selectedlibrary, the phagemid DNA was extracted and the vector was modified toremove the bacteriophage coat protein III-encoding region of the phage.The phagemid DNAs were religated and transformed into E coli XL-1 Blueto produce soluble Fabs. The vector pComb3H was constructed to encode asix-histidine tail at the end of the Fab fragment, thus facilitating Fabpurification. Fab4 purity was determined by SDS-PAGE, followed bycolloidal Coomassie brilliant blue staining

Production and Purification of HEV VLPs

The production and purification of HEV VLPs were conducted as described(Li et al., 2005, J. Virol. 79:12999-13006; Li et al., 1997, J Virol71:7207-7213; Niikura et al., 2002, Virology 293:273-280; Xing et al.,1999, Virology 265:35-45). Briefly, DNA fragments encoding theN-truncated ORF2 protein (for wild type VLPs) and the chimeric ORF2protein (for VLPs/C-tag) were cloned using the baculovirus transfervector pVL1393 to yield pVLORF2. Insect Sf9 cells (Riken Cell Bank,Tsukuba, Japan) were used to produce recombinant baculovirus. Tn5 insectcells were infected with the recombinant baculoviruses at a multiplicityof infection of 5 and incubated in EX-CELL™ 405 medium (JRH Biosciences,Lenexa, Kans.) for 6 days at 26.5° C. The supernatant was collectedafter removal of cell debris by centrifugation at 10,000 g for 90 min.The HEV VLPs were pelleted at 100,000 g for 2 h in a Beckman SW32 Tirotor and resuspended in 4.5 ml EX-CELL™ 405. The VLPs were furtherpurified by centrifugation through a CsCl density gradient (1.31 g/ml)at 110,000g for 24 h at 4° C. in a Beckman SW55Ti rotor. The white virusband was collected and diluted 4 times with EX-CELL™ 405 to decrease theCsCl concentration, and then the VLPs were centrifuged for 2 h in aBeckman TLA 55 rotor at 100,000 g. The VLPs were re-suspended in 100-500μl of 10 mM potassium-MES buffer (pH=6.2) and stored at 4° C. Toconstruct chimeric VLPs/C-tag, recombinant baculoviruses were preparedby inserting the B-cell tag epitope from herpes simplex virusglycoprotein D (QPELAPEDPED) at amino-acid position 608 (Niikura et al.,2002, supra).

Western Blotting

A series of DNA fragments were constructed to encode truncated ORF2residues 112-660, 112-608, 112-602, and 112-601, 112-600, 112-596, and112-589, respectively. These recombinant ORF2 genes were inserted into abaculovirus vector and expressed in insect cells using the protocol forVLP production, except that the recombinant proteins were recovered fromcytoplasm after lysis of the cell. Recombinant proteins were heated in4× Laemelli sample buffer and electrophoresed under reducing conditionsin a 10% SDS-polyacrylamide gel. After transfer of proteins to a PVDFmembrane, the membrane was blocked with TBS buffer (20 mM Tris, pH=7.6,NaCl) containing 0.5% Tween-20 (v/v) prior to overnight incubation withFab224 fragments at a 1:10 dilution. After extensive wash with TBSbuffer containing 0.05% Tween-20 (v/v), alkaline-phosphatase conjugatedanti-mouse IgG (Fab specific) was incubated with membrane for 1 hour atroom temperature. The blot was then washed and developed with theNBT-BCIP reaction.

Preparation of VLP-Fab Complexes for Cryo-electron Microscopy

The VLP/Fab complexes were prepared by incubating Fabs with VLPs at amolar ratio exceeding 1:300 (VLP vs Fabs) at 4° C. overnight. To reducethe background density in the subsequent structural determination,highly pure VLP/Fab complexes were obtained using a short columncontaining Sephacryl-300, which resulted in the removal of the unboundFab from the sample. The fractions containing VLP/Fab complexes werecollected based on their optical density readings at a wavelength of 280nm. The Fab binding occupancy was roughly estimated by performingSDS-PAGE (gradient 8-25%) on the purified VLP/Fab complexes at aconstant voltage using the Phast™ system (Pharmacia). The particlemorphology of VLP/Fab complexes was examined by negative stain electronmicroscopy (EM) using 2% uranyl acetate.

Cryo-electron Microscopy

Sample preparation and cryo-EM were performed following previouslydescribed, wellestablished procedures (Li et al., 2005, J. Virol.79:12999-13006; Xing et al., 1999, Virology 265:35-45). Briefly, a dropcontaining 3.5 μl of the sample was applied to a glow-dischargedholey-carbon coated copper grid, blotted with a piece of filter paperfor 3 s to remove the extra liquid and quickly plunged into liquidethane cooled by liquid nitrogen. Samples were frozen in a thin layer ofvitrified ice. The grid was then transferred into a Gatan 626DHcryo-holder and kept at a low temperature (−178° C.) during thesubsequent data collection. Micrographs were collected under a low-dosecondition (<10 e-/Å2) using Kodak SO163 film at a magnification of45,000× on a FEI CM-120 electron microscope operated at 120 kV, andparticles were photographed at a defocus range from 1000 to 3000 nm.Micrographs were visually inspected and selected based on a suitableparticle concentration, optimal ice thickness, and minimal specimendrift. Only micrographs fulfilling these criteria were analyzed.

Image Processing

Selected micrographs were digitized using a Heidelberg Prime scannerD8200 (Heidelberg, Germany) at a 14 μm scanning step size, correspondingto 3.11 Å per pixel of specimen space. Particles were manually pickedand centered by cross-correlating each one against the circular averageimage. The astigmatism and defocus value were evaluated by thesuperimposed power spectra from all particles within a singlemicrograph. The CTF first zero was approximately within the range of17-20 Å⁻¹ for the data used for the structural determination. Theself-common-lines algorithm (Crowther, 1971, Philos Trans R Soc Lond BBiol Sci 261:221-230) was used to yield the initial models forVLP/C-tag, VLP/Fab4, and VLP/Fab224. The origins and orientations searchfor each particle was carried out iteratively using the polar Fouriertransformation (PFT) algorithm running on an AMD MP1800 MHzdual-processor Linux workstation (Baker and Cheng, 1996, J. Struct.Biol. 116:120-130). Three-dimensional reconstructions were computed bycombining a set of particles with orientations that spread evenly in anicosahedral asymmetric unit using the Fourier-Bessel algorithm and bysuperimposing 5-3-2 icosahedral symmetry. To examine the reliability ofthe three-dimensional reconstruction, the dataset was evenly dividedinto two parts at the final refinement step and two three-dimensionalreconstructions were computed. The resolution was estimated usingFourier shell correlation (FSC) by assessing the agreement between thesetwo reconstructions in Fourier space. Using a coefficient value of 0.5as the criteria, the estimated resolution of the three-dimensionalreconstructions of VLP/C-tag, VLP/Fab224 and VLP/Fab4 were computed as17.5 Å, 18.5 Å and 24 Å, respectively.

The three-dimensional reconstructions were rendered and visualized usingthe Chimera program (Pettersen et al., 2004, J Comput Chem.25:1605-1612). The contour level was chosen at a value corresponding to100% of the mass of the PORF2 protein. The electron density map wasdisplayed in the isosurface mode, which builds a barrier to contour thedensity about a certain threshold.

Fitting the Crystal Structure into Cryo-EM Density Maps

Density of the bound Fab molecule was determined from a differencedensity map, which was calculated by subtracting the cryo-EM map ofunbound HEV T=1 VLP from the density map of the Fab-VLP complex. Thecryo-EM map of unbound HEV VLP was published previously (Xing et al.,1999, Virology 265:35-45). Because the cryo-EM data of unbound VLP andFab-VLP complex were collected with the same FEI CM-120 electronmicroscope under similar imaging conditions, the difference density mapwas calculated by direct subtraction of the density of unbound VLP fromthe reconstruction of Fab-VLP complex after normalizing the contrastbetween the two maps. The calculated difference map was used asconstraint in model fitting. Manual fitting was carried out bytranslational and rotational movement of the three-dimensional crystalstructure of PORF2 protein (PDB code: 2ZZQ) (Xing et al., 2010, J BiolChem 285:33175-33183) into the cryo-EM density maps using program 0(Jones et al., 1991, Acta crystallogr. Sect. A 47:110-119). To obtainthe best fit, the atomic model of the PORF2 subunit was treated as arigid body. The fitting was first manually refined by minimizing thecrashes between symmetry-related PORF2 molecules and then evaluatedbased on the cross correlation coefficient (cc value) between thecryo-EM density and the density computed from the fitted PORF2coordinates. Fitting was halted when the cc value reached 80%. Thefigures were prepared using the program PyMOL (DeLano, 2002, The PYMOLmolecular graphics system. DeLano Scientific, Palo Alto, Calif., USA),and the surface stereographic projection of the HEV VLP was preparedusing the program RIVEM (Xiao and Rossmann, 2007, J Struct. Biol.158:181-186).

Results

Binding of Antibody Mab224 to PORF2

Binding of the monoclonal antibody Fab224 to PORF2 was examined viaimmunoblot analysis. A series of recombinant ORF2 proteins withC-terminal truncations were separated by SDS-PAGE on a 10% gel underreducing conditions and blotted with Fab224 (FIG. 35A). Fab224recognized both reduced and denatured recombinant ORF2 proteins thatcontained amino acids 112-660, 112-608, 112-602, and 112-601. Incontrast, recombinant ORF2 proteins composed of residues 112-600,112-596, and 112-589 did not bind to Fab224. These data indicate thatresidues 597-601 are critical for Fab224 binding to PORF2. Because therecombinant ORF2 proteins were recovered from cell cytoplasm wheremultiple forms of PORF2 were reported (Li et al., 1997, J Virol71:7207-7213), the positive bands observed at low molecular weight maybe the proteolytic products or degraded forms of ORF2 that contain theFab224 binding sequence.

Two-dimensional Electron Cryo-micrographs

The chimeric VLPs (FIG. 35C) and the Fab224-conjugated VLP complex (FIG.35D) showed circular profiles with spike-like densities that extendedfrom the surface. As observed previously (Li et al., 1997, supra; Xinget al., 1999, Virology 265:35-45), they appeared to have a whitecontrast center, indicating that they are empty particles lacking RNA(data not shown). The sizes of both VLPs were approximately 27 nmwithout taking into account the extra densities that extended from theVLP/Fab224 surface (FIG. 35D).

Binding Site of Antibodies

The cryo-EM structure of HEV/Fab224 was reconstructed from 615 images ofindividual particles and displayed T=1 icosahedral symmetry with 60protein subunits that were arranged into 30 dimeric protruding spikeslocated at each icosahedral two-fold axis (FIG. 36A). Sixty Fabmolecules were observed around each VLP particle, bound to the shoulderof the P domain. The Fab density extended ˜57 Å radially away from thespike surface. The density corresponding to the Fab was approximatelyequal in magnitude to that of the HEV VLP, indicating that most or allof the 60 binding sites were occupied by a Fab molecule. The densitycorresponding to the VLP capsid was removed from the cryo-EM map,producing a Fab differential density map that was used to pinpoint thebinding site of the Fab224 antibody (FIGS. 37A and 37B).

In addition, the structure of HEV VLP in complex with the neutralizingantibody Fab4 was determined by combining 264 individual images. Fab4precipitates both the native HEV virion and recombinant PORF2 peptidesbut the reaction depends on the presence of amino acids 597-607(Schofield et al., 2003, Vaccine 22:257-267). Three-dimensionalreconstruction of the VLP/Fab4 complex showed 60 Fab molecules bound toeach HEV VLP. Unlike the Fab224 VLP complex, the density correspondingto Fab4 was about one third of that of the capsid (FIG. 36A), suggestingthat only 30-40% of the binding sites were occupied by the Fab.Moreover, the binding of Fab4 appeared deeper on the side-wall of theprotruding domain towards the capsid shell, leaving its Fc domainexposed above the surface of the plateau (FIG. 36A). In contrast, theentire Fab224 stood mainly on the top of the P domain surface. TheFab224 and the Fab4 extend along the long axis of the P domain. In bothcases, no steric hindrance of the Fab on the P domain with theneighboring Fab molecules at either the five-fold or the three-fold axeswas apparent. The orientation of the Fab relative to the plateauappeared different at a radius of 135 Å. The long axis of Fab224 tiltedtoward the neighboring spike, while the long axis of Fab4 pointed to thefivefold axis (FIG. 36A).

To further analyze the Fab and HEV VLP binding interface, the crystalstructure of genotype-1 PORF2 was docked onto the VLP/Fab224 cryo-EMdensity map. The genotype-1 PORF2 crystal structure (2ZZQ) is composedof three domains (Xing et al., 2010, J Biol Chem 285:33175-33183), andthese domains are in good agreement with those of genotype-3 andgenotype-4 PORF2 (PDB accession number 2ZTN and 3HAG, respectively) (Guuet al., 2009, supra; Yamashita et al., 2009, Proc Natl Acad Sci USA106:12986-12991). The coordinates fitted very well with the cryo-EMdensity map without any adjustment (CC value=80%). The atoms on thesurface of the HEV VLP capsid were plotted and colored according totheir radial distance, and overlapped with the density of the Fab at thesurface plateau of the protruding spike (FIG. 36B).

The Fab224 interacted with the residues at the side of the ORF2 spikerather than with those residues on the spike plateau surface (FIG. 37C).The contact footprint did not overlap with the dimeric interface of thePORF2 spike. As expected, Fab224 recognizes a conformational epitopesand its binding site covers a surface composed of three loops includingamino acids 470-493 in loop 1, amino acids 539-569 in loop 2, and aminoacids 581-595 in loop 3 (FIG. 37D). Residues E479, D481, T484, Y485, andS487 from the AB loop and residues Y532, S533, and K534 from the CD loopwere in close contact with the Fab molecule.

Structure of HEV Chimeric VLP

Chimeric HEV VLP/C-tag was constructed using a PORF2 fusion protein inwhich a B-cell tag of 11 amino acids was incorporated into theC-terminus of PORF2 (FIG. 35B). A total of 782 images of individualparticles were used to reconstruct the final three-dimensional model ofVLP/C-tag. In agreement with the previously published cryo-EM VLPstructures, the surface of VLP/C-tag can also be divided into twodistinct layers, an icosahedral shell and a protruding spike (FIG. 38A).The spike projects outward from the icosahedral shell and is composed ofa PORF2 dimer. The distance between two adjacent spikes was ˜76 Å asmeasured between the centers of surface plateau. These results areconsistent with the measurements of VLPs obtained either from Tn5 insectcells (Xing et al., 1999, Virology 265:35-45) or from Sf9 insect cells(Li et al., 2005, J. Virol. 79:12999-13006) and no detectable densitywas added onto the outer surface of the spike. No RNA density wasdetected within the chimeric VLP/C-tag.

The crystal structure fit very well within the VLP/C-tag density map(FIG. 38B), indicating that the C-terminal 11 amino acids insertioninhibits neither the dimer-dimer interactions nor the formation of T=1VLP. When the density maps were contoured to cover 100%, the radii ofthe S domains were roughly the same for both the VLP/C-tag and theVLP/Fab224 maps, and the height of the protruding spikes appearedsimilar. Density difference was not observed from the docking (FIG. 39),suggesting that the inserted B-cell tag is flexible and less ordered.However, model fitting revealed that coordinates-unoccupied densityappeared at the lateral side of the spike and underneath the Fab224binding site (FIGS. 39A and 39B), which may correspond to the insertedpeptide.

Discussion

HEV T=1 VLP is a vaccine candidate that induces protective immunity innon-human primates (Li et al., 2004, Vaccine 22:370-377). It can also beused as an antigen carrier to deliver foreign epitopes through oraladministration (Niikura et al., 2002, Virology 293:273-280). Therefore,structural analysis of the antibody recognition sites is essential tosuppress the neutralization effect of host vector-specific antibodies.For this purpose, the inventors determined the structure of HEV VLP incomplex with antibodies Fab224 (VLP/Fab224), Fab4 (VLP/Fab4), and thestructure of chimeric HEV VLP carrying a B-cell tag at the C-terminus ofPORF2 (VLP/C-tag). Docking the PORF2 crystal structure provides spatialinformation on the HEV antigenic domain and structural guidance tobetter design foreign epitope insertion.

Structure of the Neutralization Epitopes

The antigenic properties of HEV and the mechanisms by which it isneutralized are difficult to characterize due to the lack of adequatecell culture replication systems. Therefore, our understanding of HEVimmunology is mainly based on studies using recombinant proteinsexpressed in E. coli (Riddell et al., 2000, J Virol 74:8011-8017) andrecombinant proteins or HEV VLPs generated using the baculovirusexpression systems (Li et al., 1997, J Virol 71:7207-7213; Robinson etal., 1998, Protein Expr Purif 12:75-84). Data from these studiesindicates that the C-terminal region of PORF2 participates in the immuneresponse against HEV and that the HEV neutralization epitope isconformational. The minimum peptide required to induce HEV neutralizingantibodies corresponds to a region of 148 residues in PORF2, from aminoacids 459-607 (Zhou et al., 2005, Vaccine 23:3157-3165). This peptidecoincides with the P-domain revealed in the crystal structures of PORF2.The density of the Fab in our cryo-EM structure interfaced entirely withthe spikes, thus confirming that the P domain is primarily responsiblefor HEV antigenicity. Fab4 is a chimpanzee antibody that recognizes ORF2protein and was isolated using phage display from a cDNA library(Schofield, et al., 2000, J Virol 74:5548-5555). Fab4 binds to nativeHEV virions and recombinant PORF2 peptides containing amino acids597-607 (Schofield et al., 2003, Vaccine 22:257-267). The inventorsperformed fitting with the VLP/Fab4 structure; however, the Fab4 densitywas too weak to conclusively determine the Fab4 binding site on thesurface of HEV VLP. However, the density corresponding to the Fab4molecule did cover amino acid 606 (data not shown). It is not clear whyFab224 appeared not to interact with peptides lacking amino acids599-608 in immunoblot analysis. However, the Fab224 binding site isconsistent with the critical antigenic residues determined previouslyusing mutagenesis. It was found that double mutations that changedresidues E479 and K534 or Y485 and 1529 to alanine selectively abrogatedPORF2 reactivity with neutralizing antibodies (Li et al., 2009, PLosPathog. 5:e1000537). Another set of mutants defined the same region asthe HEV antigenic domain, with antibody recognition residues spreadingover the AB, CD and EF loops (Yamashita et al., 2009, Proc Natl Acad SciUSA 106:12986-12991). The antibodies used in both experiments wereneutralizing antibodies; therefore, the Fab224 binding surface is partof the dominant neutralization site, suggesting that the monoclonalantibody Fab224 is a neutralizing antibody. This neutralization sitepartially overlaps with the receptor binding site (Yamashita et al.,2009, supra), and antibody binding may create spatial hindrance thatprevents HEV VLPs from attaching to the cell surface.

Insertion Sites for Foreign Epitopes

Because they are highly organized capsids that mimic the overallstructure of virus particles, VLPs are a robust means by which tosimultaneously carry small molecules, peptide antigenic epitopes, andDNA vaccines from heterogeneous sources to target disease sites.However, this rational vaccine design relies on excellent VLP structuralinformation so that epitopes can be effectively conjugated to the VLPsurface. In previous study, rather than selecting PORF2 insertion siteson the basis of structural information, six insertion sites wereselected according to restriction enzyme sites located either internally(four sites) or in the N- or C-terminus of PORF2. The internal sites arelocated after residues A179, R366, A507, and R542. Fusion proteinscarrying insertions at sites A179 and R336 failed completely to produceVLPs, and insertions at A507 and R542 greatly reduced VLP production(Niikura et al., 2002, supra). Crystal structure data revealed that thespatial position of these sites is disadvantageous. Residue A179 islocated in the S domain in the middle of a a-helix, which is necessaryfor the integrity of the S domain and its interaction with the twofoldrelated neighboring subunit. R366 is located in the M-domain and favorselectrostatic interaction with residue E386 from the threefold relatedneighboring subunit. Although located within the P domain, the sidechain of R542 is within the dimeric interface and guides the hydrophobicinteraction of the two monomers. Replacement of R542 may misalign theorientation between two P domains and weaken the dimeric interactionbetween PORF2 proteins. Residue A507 in the P-domain plays an importantrole in maintaining P domain orientation by fixing the angle of the longproline-rich hinge. Moreover, none of the four residues are exposed onthe surface of VLPs, although some of them are located on the surface ofindividual PORF2 subunits (FIGS. 38C and 38D). Therefore, insertion of aforeign sequence at these sites does not interfere with the expressionof individual proteins but rather hinders the assembly of HEV VLPs. Thecrystal structure revealed that the C-terminus is exposed on the surfaceof VLPs, while the N-terminus points toward the VLP center. Therefore,insertion at these two sites does not inhibit VLP assembly; however, theC-terminus is more suitable for tethering bulky foreign antigenicsequences, as was shown in a previous report (Niikura et al., 2002,supra).

The cryo-EM structure of the chimeric HEV VLP/C-tag suggested that theB-cell tag was located at the lateral side of the spike, not far fromresidue A606 (C-terminal end in the crystal structure) (FIG. 39A). Thisdensity is located beneath the Fab224 binding site but nonethelessoverlaps with the potential binding site of Fab4. As a result, insertionof the 11 amino acid B-cell sequence may partially leave the HEVantigenic site open and accessible to the host immune system. Thisexplains why mice can develop antibodies against both HEV and theforeign epitope after VLP/C-tag oral administration (Niikura et al.,2002, supra).

In conclusion, the cryo-EM structures of VLP/Fab224 identified thelateral surface of the P domain as the recognition site for anti-HEVneutralizing antibodies. Insertion of a B-cell epitope at the PORF2C-terminus does not interfere with T=1 VLP assembly. Thus, T=1 HEV VLPsare a novel tool for oral vaccine delivery, as they constitute anon-replicating entities that can induce mucosal immunity withoutadjuvant. Induction of antibodies against both HEV and the targetdisease is an additional advantage of this delivery system.

All patents, patent applications, and other publications, includingGenBank Accession Numbers, cited in this application are incorporated byreference in the entirety for all purposes.

What is claimed is:
 1. A composition comprising: an HEV capsid proteincomprising a portion of an HEV ORF 2 protein and a heterologous peptide,wherein the heterologous peptide is inserted into the portion of HEV ORF2 within a pre-selected region corresponding to residues 483-490,residues 530-535, residues 554-561, residues 573-577, or residues582-593, or residues 601-613 of SEQ ID NO:1; and a heterologous nucleicacid encapsulated in a chimeric HEV virus-like particle (VLP) formed bythe HEV capsid protein.
 2. The composition of claim 1, wherein theheterologous nucleic acid and the heterologous peptide are from the samesource.
 3. The composition of claim 1, wherein the heterologous nucleicacid and the heterologous peptide are from different sources.
 4. Thecomposition of claim 1, wherein the HEV capsid protein comprises aportion of HEV ORF 2 protein and two or more heterologous peptides. 5.The composition of claim 1, wherein the two or more heterologouspeptides are from different sources.
 6. The composition of claim 1,wherein the two or more heterologous peptides are from the same source.7. The composition of claim 1, wherein the HEV capsid protein comprisesan amino acid sequence corresponding to residues 112-608 of SEQ ID NO:1.8. The composition of claim 7, wherein the HEV capsid protein furthercomprises an HEV ORF3 polypeptide corresponding to residues 91-123 ofSEQ ID NO:2 fused to the N terminus of an HEV ORF2 polypeptidecorresponding to residues 112-608 of SEQ ID NO:1.
 9. The composition ofclaim 7, wherein the HEV capsid protein further comprises an HEV ORF3polypeptide corresponding to residues 70-123 of SEQ ID NO:2 fused to theN terminus of an HEV ORF2 polypeptide corresponding to residues 112-608of SEQ ID NO:1.
 10. A composition of claim 1, further comprising apharmaceutically acceptable excipient.
 11. A composition of claim 10,wherein the excipient is an adjuvant.
 12. A composition of claim 10,wherein the excipient is adapted for oral delivery.
 13. A composition ofclaim 10, wherein the excipient is adapted for mucosal delivery.
 14. Amethod of inducing an immunogenic response in a host, comprising thestep of administering the composition of claim
 1. 15. An HEV capsidprotein, comprising a portion of an HEV ORF 2 protein and a heterologouspeptide inserted into the portion of the HEV ORF 2 within a pre-selectedregion of residues corresponding to residues 483-490, residues 530-535,residues 554-561, residues 573-577, residues 582-593, or residues601-613 of SEQ ID NO:1, wherein the HEV capsid protein forms an HEV VLP.16. A polynucleotide encoding the HEV capsid protein of claim 15.